NOS mRNA Research Articles

Interferon‐γ regulates chemokine expression and release in the human mast cell line HMC1: role of nitric oxide

Mast cells (MCs) are critical immune effector cells that release cytokines and chemokines involved in both homeostasis and disease. Interferon-gamma (IFN-gamma) is a pleiotropic cytokine that regulates multiple cellular activities. IFN-gamma modulates rodent MC responsiveness via production of nitric oxide (NO), although the effects in human MC populations is unknown. We sought to investigate the effects of IFN-gamma on expression of the chemokines interleukin-8 (IL-8) and CCL1 (I-309) in a human mast cell line (HMC1) and to determine the underlying regulatory mechanism. Nitric oxide synthase (NOS), IL-8 and CCL1 expression was determined using real-time polymerase chain reaction (PCR). NOS protein expression was analysed using western blot. NOS activity was determined using the citrulline assay. IL-8 and CCL1 release was measured by specific enzyme-linked immunosorbent assay (ELISA). IFN-gamma inhibited phorbol 12-myristate 13-acetate (PMA)-induced release of IL-8 and CCL1 (by 47 and 38%). Real-time PCR analysis of IFN-gamma-treated HMC1 showed a significant (P < 0.05) time-dependent increase in NOS1 and NOS3 mRNA. NOS3 protein was significantly increased at 18 hr, which correlated with a significant (P < 0.05) increase in constitutive NOS (cNOS) activity. IFN-gamma-induced inhibition of chemokine expression and release was NO dependent, as treatment with the NOS inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) reduced the IFN-gamma inhibitory effect on IL-8 and CCL1 mRNA expression. NO donors mimicked the IFN-gamma effect. IFN-gamma inhibited PMA-induced cAMP response element binding protein (CREB) phosphorylation and DNA-binding activity. Our observations indicate for the first time that IFN-gamma enhances endogenous NO formation through NOS3 activity, and that NO regulates the transcription and release of IL-8 and CCL1 in a human MC line.

Impact of foetus and mother on IFN-γ-induced indoleamine 2,3-dioxygenase and inducible nitric oxide synthase expression in murine placenta following Toxoplasma gondii infection

IFN-γ production is a hallmark of acute infection with the protozoan parasite Toxoplasma gondii. The tryptophan-catabolising enzyme indoleamine 2,3-dioxygenase (IDO), as well as inducible nitric oxide synthase (NOS2) are induced by IFN-γ and can play extremely diverse roles in immune regulation, defence against pathogens and physiological homeostasis. We investigated the regulation of these two central enzymes in the placenta during acute infection of pregnant female mice. Using IFN-γ receptor knockout (IFNγR −/−) mice, we showed that IDO is not constitutively expressed in term placentas. In contrast, NOS2 expression was observed, largely dependent on IFN-γ signalling. Upon infection with the avirulent PRU strain of T. gondii, IDO mRNA expression was induced in an IFNγR-dependent manner. Surprisingly, NOS2 mRNA was severely suppressed. Importantly, we showed in crossing experiments of heterozygote (IFNγR +/−) mothers with IFNγR −/− males and vice versa that IDO expression largely depends on the presence of IFN-γ receptors on foetal cells, and to a lesser extent on maternal cells. Immunohistochemical analysis localised foetal IDO production to invasive trophoblasts within the maternal part of the placenta. The placental vascular endothelium only stained positive when the mothers possessed functional IFN-γ receptors. In contrast, placental NOS2 expression, but also its suppression following infection, seems to be largely dependent on IFN-γ signalling in maternal cells. Neither factor appears to regulate placental T. gondii growth, as we observed no difference in parasite numbers between (+/−) and (−/−) foetuses. Taken together, our results demonstrate the crucial role of the foetus in placental IDO, but not NOS2, production following T. gondii infection.

Molecular Basis of RNA Recognition by the Embryonic Polarity Determinant MEX-5

Embryonic development requires maternal proteins and RNA. In Caenorhabditis elegans, a gradient of CCCH tandem zinc finger (TZF) proteins coordinates axis polarization and germline differentiation. These proteins govern expression from maternal mRNAs by an unknown mechanism. Here we show that the TZF protein MEX-5, a primary anterior determinant, is an RNA-binding protein that recognizes linear RNA sequences with high affinity but low specificity. The minimal binding site is a tract of six or more uridines within a 9-13-nucleotide window. This sequence is remarkably abundant in the 3'-untranslated region of C. elegans transcripts, demonstrating that MEX-5 alone cannot specify mRNA target selection. In contrast, human TZF homologs tristetraprolin and ERF-2 bind with high specificity to UUAUUUAUU elements. We show that mutation of a single amino acid in each MEX-5 zinc finger confers tristetraprolin-like specificity to this protein. We propose that divergence of this discriminator residue modulates the RNA-binding specificity in this protein class. This residue is variable in nematode TZF proteins, but is invariant in other metazoans. Therefore, the divergence of TZF proteins and their critical role in early development is likely a nematode-specific adaptation.

Inhibition of translation initiation by endotoxin and interferon gamma in skeletal muscle cells is nitric oxide synthase (NOS) dependent

Excess production of inflammatory mediators during the innate immune response can induce tissue damage, sepsis and shock. Among these mediators nitric oxide (NO) is bactericidal for microorganisms and yet suicidal for the host when in excess. Inflammation inhibits protein synthesis in skeletal muscle but the affect of excess NO on protein synthesis is unknown. The purpose of the present study was to test the hypothesis that endogenous NO negatively affects translation initiation and elongation in C2C12 myotubes. LPS and IFN individually had no affect on protein synthesis but in combination they inhibited protein synthesis by 80%. The combination of LPS and IFN dramatically down regulated the auto-phosphorylation of mTOR and its substrates S6K1 and 4EBP-1. The phosphorylation of ribosomal protein S6 was decreased whereas phosphorylation of elongation factor-2 (eEF-2) was enhanced consistent with defects in both initiation and elongation. Loss of S6 phosphorylation occurred 8–12 h after the addition of LPS and IFN and coincided with a prolonged expression of NOS2 mRNA and protein. Pre-treatment of myotubes with L-NAME prevented the LPS/IFN-induced decrease in protein synthesis and restored signaling downstream of mTOR. The results suggest that LPS and IFN induce NOS-2 which generates excess NO that consequently has a negative affect on translation initiation and elongation in skeletal muscle. (GM38032)

Reduced neuronal nitric oxide synthase expression contributes to cardiac oxidative stress and nitroso-redox imbalance in ob/ob mice

Disruption of leptin signaling in the heart may contribute to obesity-related cardiac disease, as leptin deficient ( ob/ob) mice display cardiac hypertrophy, increased cardiac apoptosis and reduced survival. Since leptin maintains a tonic level of neuronal nitric oxide synthase (NOS1) expression in the brain, we hypothesized that leptin deficiency would decrease NOS1 cardiac expression, in turn activating xanthine oxidoreductase (XOR) and creating nitroso-redox imbalance. We studied 2- to 6-month-old ob/ob ( n = 26) and C57Bl/6 controls ( n = 27). Cardiac NOS1 protein abundance ( P < 0.01) and mRNA expression ( P = 0.03) were reduced in ob/ob ( n = 10 and 6, respectively), while NOS3 protein abundance and mRNA expression were unaltered. Importantly, cardiac NOS1 protein abundance was restored towards normal in ob/ob mice after leptin treatment ( n = 3; P < 0.05 vs leptin untreated ob/ob mice). NO metabolite (nitrite and nitrate) production within the myocardium was also reduced in ob/ob mice ( n = 5; P = 0.02). Furthermore, oxidative stress was increased in ob/ob mice as GSH/GSSG ratio was decreased ( n = 4; P = 0.02). Whereas XOR activity measured by Amplex Red fluorescence was increased ( n = 8; P = 0.04), XOR and NADPH oxidase subunits protein abundance were not changed in ob/ob mice ( n = 6). Leptin deficiency did not disrupt NOS1 subcellular localization, as NOS1 co-localized with ryanodine receptor but not with caveolin-3. In conclusion, leptin deficiency is linked to decreased cardiac expression of NOS1 and NO production, with a concomitant increase in XOR activity and oxidative stress, resulting in nitroso-redox imbalance. These data offer novel insights into potential mechanisms of myocardial dysfunction in obesity.

Interleukin-1β Signals through a c-Jun N-Terminal Kinase-Dependent Inducible Nitric Oxide Synthase and Nitric Oxide Production Pathway in Sertoli Epithelial Cells

Our recent Sertoli cell (SC) studies showed that the c-Jun N-terminal kinase (JNK) and inducible cyclooxygenase-2 (COX-2) pathways are key regulatory components of IL (IL-1alpha, IL-1beta, and IL-6) expression and START-domain containing StARD1 and StARD5 proteins. IL-1beta regulates SC autocrine/paracrine activities and subsequently influences developing germ cells and spermatogenesis. This study was designed to evaluate whether IL-1beta mediates high-output inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production in these specialized epithelial cells and characterize gonadotropin and cytokine-regulation of NO. Purified SCs were maintained in serum-free cultures and treated with FSH (100 ng-1 microg/ml) or IL-1beta (10 ng/ml) in time-course studies. To determine obligatory intracellular pathways, treatments were conducted with or without activity inhibitors: COX-2 selective (NS-398, 10 microM) or JNK (SP600125, 10 microM) for 1, 3, 6, and 24 h. NOS mRNAs and proteins were evaluated by RT-PCR and Western analysis, respectively. NO and reactive oxygen species were measured by flow cytometry and ELISA. IL-1beta transiently induces intracellular NO (30 min) but not reactive oxygen species. Subsequently, iNOS mRNA and protein expression (3-6 h) significantly increased after IL-1beta but not FSH stimulation, and in time-dependent manner, markedly increased extracellular NO (24 h, 8-fold). No change in the constitutive endothelial NOS isoform was observed. Inhibition of JNK, but not COX-2, activity inhibits IL-1beta-induced iNOS expression and NO production. Such findings suggest that intra- and extracellular NO within the tubule may alert SCs monitoring the microenvironment to an aberrant cytokine, triggering antioxidant and antiinflammatory activities to avoid disruption of spermatogenesis.

In vivo protective effect of melatonin on cadmium‐induced changes in redox balance and gene expression in rat hypothalamus and anterior pituitary

Cadmium (Cd) is widely used in industrial applications and is an important side contaminant of agricultural products. As an endocrine disruptor, Cd modifies pituitary hormone release. It has been shown that this metal causes oxidative stress in primary cultures of anterior pituitary cells. To examine whether Cd induces redox damage in the hypothalamic-pituitary axis in vivo and to evaluate the efficacy of the antioxidant molecule melatonin to prevent Cd activity, rats were exposed to Cd (5 p.p.m. in drinking water) with or without melatonin (3 microg/mL drinking water) for 1 month. In the anterior pituitary, Cd increased lipid peroxidation and mRNA levels for heme oxygenase-1 (HO-1) at both time intervals tested (09:00 and 01:00 hr, beginning of rest span and middle of activity span, respectively). Melatonin administration prevented the Cd-induced increase in both parameters. In the hypothalamus, Cd affected the levels of mRNA for HO-1 by decreasing it in the evening. Melatonin reduced hypothalamic HO-1 gene expression. Cd treatment augmented gene expression of nitric oxide synthase (NOS)1 and NOS2 in the pituitary whereas melatonin decreased it, impairing the activity of Cd. Exposure to Cd increased the levels of hypothalamic NOS1 mRNA at 09:00 hr and decreased the levels of NOS2 mRNA at 01:00 hr, with melatonin treatment preventing Cd effects. Cd treatment decreased plasma thyroid-stimulating hormone levels at both examined times, while melatonin reversed the effect of Cd at 09:00 hr and partially counteracted the effect at 01:00 hr. There were important variations between day and night in the expression of all the genes tested in both tissues. Melatonin treatment was effective reducing all examined effects of Cd, documenting its effectiveness to protect the rat hypothalamic-pituitary axis from the toxic metal effects.

Interrelated overexpression of endothelial and inducible nitric oxide synthases, endothelin‐1 and angiogenic factors in human papillary thyroid carcinoma

Nitric oxide (NO) and endothelin-1 (ET-1) are involved in carcinogenesis. Overexpression of the ET-1 axis has been demonstrated in papillary thyroid carcinoma (PTC). This study investigated the expression of NO synthases (NOS) and their relationship with expression of ET-1 and angiogenic markers in PTC. Expression of NOS, angiogenic markers [vascular endothelial growth factor (VEGF), angiopoietin-1 and angiopoietin-2] and their receptors was studied in surgical thyroid samples obtained from 22 patients aged 15-68 years. Three groups were constituted: normal thyroid (n = 5), Hashimoto's thyroiditis (n = 9) and PTC (n = 8). Immunohistochemistry disclosed NOS2 and NOS3 immunoreactivity in PTC cells, the percentage of positive cells being greater than normal (P < 0.02). Real-time quantitative polymerase chain reaction (RTQ-PCR) showed that NOS2 and NOS3 mRNA levels were, respectively, increased (P < 0.02) by 2.6 +/- 0.6 and 4.2 +/- 1.1 times in PTC. RTQ-PCR demonstrated that VEGF, its receptors VEGFR-1 and VEGFR-2, and angiopoietin-2 and its receptor (Tie2) were also overexpressed (P < 0.05) in PTC. Correlations were found between ET-1 expression and that of NOS2, angiopoietin-1 and -2 (P < 0.05). NOS2 mRNA levels also correlated with those of NOS3 and angiopoietin-2 (P < 0.05). In thyroiditis, NOS2 immunoreactivity was observed in inflammatory cells whereas NOS2 mRNA levels were 12.1 +/- 1.6 times higher than normal (P < 0.005). This study revealed an activation of the NO pathway in thyroid carcinoma, which is interrelated to the ET-1 axis, both systems being overexpressed in concert with angiogenic factors. This global system might play a role in carcinogenesis and constitutes a potential target for anticancer therapy.

Characterization of NO and Cytokine Production in Immune-Activated Microglia and Peritoneal Macrophages Derived from a Mouse Model Expressing the Human NOS2 Gene on a Mouse NOS2 Knockout Background

Significant differences exist in the production and release of nitric oxide (NO) from human macrophages versus macrophages of mouse origin. Human macrophages have been shown to respond poorly to stimuli that provoke strong inflammatory reactions from mouse macrophages. To address the differences in macrophage function in an animal model, a transgenic mouse was created that contained the entire human NOS2 gene, including the human promoter and all of its exons and introns. The huNOS2 transgenic mouse was then mated to mice lacking a functional NOS2 gene (muNOS2(/) or NOS2 knockout mice) to generate a double transgenic mouse (huNOS2(+/0)/muNOS2(/)) that expresses a functional human NOS2 gene in place of the mouse NOS2 gene. These double transgenic mice were found to express only human NOS2 mRNA and human iNOS proteins in response to immune stimulation. The production and release of nitric oxide from isolated macrophages from the doubly transgenic mouse also more closely paralleled human responses rather than mouse. Peritoneal macrophages from double transgenic mice generated nanomolar levels of nitrite in response to inflammatory stimuli, while peritoneal macrophages from wild-type mice generated micromolar levels of nitrite in response to the same inflammatory stimuli. Similarly, microglia from the huNOS2(+/0)/muNOS2(/) mice accumulated nanomolar levels of nitrite following inflammatory stimulation. Reduced nitrite release persisted in spite of normal responsiveness to inflammatory stimulation as measured by tumor necrosis factor alpha and interleukin-6 production and release. These data suggest that the human-specific release of nanomolar levels of nitrite may largely result from differences between the human and mouse NOS2 genes, which may program different degrees of nitric oxide responses to inflammatory signals in humans than in mice.

Effects of Noradrenaline on Neuronal NOS2 Expression and Viability

The authors previously showed that conditioned media (CM) from activated microglia increased inducible nitric oxide synthase (NOS2) in cortical neurons. Here they examined the ability of noradrenaline (NA) to reduce neuronal NOS2 or cell death. Primary mouse cortical neurons were activated using CM from microglia incubated with lipopolysaccharide (LPS). Neuronal NOS2 was assessed by increases in nitrite accumulation, and increases in NOS2 mRNA levels and fluorescence of the NO-sensitive probe DAF-2 DA. NOS2 induction was associated with an increase in neuronal LDH release. When NA was added during microglial activation, neuronal NOS2 was significantly reduced (by approximately 70%); in contrast if NA was added to the neurons along with CM, there was less reduction (about 35% decrease) in NOS2 expression. NA added to either microglia or to neurons reduced neuronal LDH release comparably. Pretreatment of CM with blocking antibody to TNFalpha, alone or with IL1-receptor antagonist, partially reduced neuronal cell death and NOS2. Incubation of neurons with NA increased IkBalpha, which could reduce NOS2. These results demonstrate that NA modulates neuronal NOS2 expression and damage, and that these effects are primarily due to inhibition of microglia released factors. Perturbations of NA could exacerbate neuronal damage by allowing for increased inflammatory responses.

High-Flow-Induced Arterial Remodeling in Rats with Different Susceptibilities to Cerebral Aneurysms

Background: The higher incidence of cerebral aneurysms (CAs) induced by enhanced arterial blood flow in Long Evans (LE) compared to Brown Norway (BN) rats suggests that intrinsic differences in high-flow arterial remodeling may be involved in determining CA susceptibility. Some aspects of this remodeling were compared in LE and BN rats after creation of an abdominal aortocaval fistula (ACF). Methods and Results: At 4 days with ACF, aortic luminal cross-sectional area (LCSA) determined by morphometry was increased by 20% in LE but not in BN rats. mRNA levels, determined by RT-PCR, were higher in LE than in BN rats for collagen α1(I), collagen α1(III), MMP2 and its inhibitor TIMP1 at 19 days with ACF. Nitric oxide synthase (NOS) mRNA levels were higher in LE rats at 4 days for the inducible (NOS2) isoform and at 4 and 19 days for the neuronal (NOS1) isoform. Aortic LCSA and NOS1 mRNA levels were tightly correlated and NOS inhibition prevented ACF-induced aortic remodeling in the LE rat. MMP2 and MMP7 activity, evaluated by zymography at 4 days with ACF, did not greatly differ between BN and LE. Conclusions: These data suggest that a higher intrinsic ability for high-flow-induced arterial enlargement associated with NOS gene overexpression may be a possible genetic determinant in CA susceptibility.

Transcription of human neuronal nitric oxide synthase mRNAs derived from different first exons is partly controlled by exon 1-specific promoter sequences

The human neuronal nitric oxide synthase ( NOS1) gene is subject to extensive splicing. A total of 12 NOS1 mRNA species have been identified. They differ in their 5′ ends and are derived from 12 different first exons (termed exons 1a to 1l). Various cell lines whose NOS1 first exon expression patterns were representative of human brain, skin, and skeletal muscle were identified. These included A673 neuroepithelioma cells, SK-N-MC neuroblastoma cells, HaCaT keratinocyte-like cells, and C2C12 myocyte-like cells. In these cell lines, correlations were found between the exon 1 variants preferentially expressed and the promoter activities of their cognate 5′ flanking sequences. These data demonstrate that expression of the different exon 1-related splice variants of NOS1 mRNA is controlled directly (at least in part) by the associated 5′ flanking sequences.

Visualization of primordial germ cells in vivo using GFP-nos1 3'UTR mRNA

In some teleost fish, primordial germ cells (PGCs) inherit specific maternal cytoplasmic factors such as vasa and nanos 1 (nos1) mRNA. It has been shown that the 3'untranslated regions (UTRs) of vasa and nos1 have critical roles for stabilization of these RNAs in zebrafish PGCs. In this study, to determine whether this role of the nos 1 3'UTR is conserved between teleost species, we injected artificially synthesized mRNA, combining green fluorescent protein (GFP) and the zebrafish nos 1 3'UTR (GFP-nos 1 3'UTR mRNA), into the fertilized eggs of various fish species. The 3'UTR of the Oryzias latipes vasa homologue (olvas ) mRNA was assayed in the same manner. We demonstrate that the PGCs of seven teleost species could be visualized using GFP-nos 1 3'UTR mRNA. GFP-olvas 3'UTR mRNA did not identify PGCs in herring or loach embryos, but did enable visualization of the PGCs in medaka embryos. Our results indicate that the 3'UTR of the zebrafish nos1 mRNA can promote maintenance of RNAs in the PGCs of different fish species. Finally, we describe and compare the migration routes of PGCs in seven teleost species.

Expression profiles for macrophage alternative activation genes in AD and in mouse models of AD

BackgroundMicroglia are associated with neuritic plaques in Alzheimer disease (AD) and serve as a primary component of the innate immune response in the brain. Neuritic plaques are fibrous deposits composed of the amyloid beta-peptide fragments (Abeta) of the amyloid precursor protein (APP). Numerous studies have shown that the immune cells in the vicinity of amyloid deposits in AD express mRNA and proteins for pro-inflammatory cytokines, leading to the hypothesis that microglia demonstrate classical (Th-1) immune activation in AD. Nonetheless, the complex role of microglial activation has yet to be fully explored since recent studies show that peripheral macrophages enter an "alternative" activation state.MethodsTo study alternative activation of microglia, we used quantitative RT-PCR to identify genes associated with alternative activation in microglia, including arginase I (AGI), mannose receptor (MRC1), found in inflammatory zone 1 (FIZZ1), and chitinase 3-like 3 (YM1).ResultsOur findings confirmed that treatment of microglia with anti-inflammatory cytokines such as IL-4 and IL-13 induces a gene profile typical of alternative activation similar to that previously observed in peripheral macrophages. We then used this gene expression profile to examine two mouse models of AD, the APPsw (Tg-2576) and Tg-SwDI, models for amyloid deposition and for cerebral amyloid angiopathy (CAA) respectively. AGI, MRC1 and YM1 mRNA levels were significantly increased in the Tg-2576 mouse brains compared to age-matched controls while TNFα and NOS2 mRNA levels, genes commonly associated with classical activation, increased or did not change, respectively. Only TNFα mRNA increased in the Tg-SwDI mouse brain. Alternative activation genes were also identified in brain samples from individuals with AD and were compared to age-matched control individuals. In AD brain, mRNAs for TNFα, AGI, MRC1 and the chitinase-3 like 1 and 2 genes (CHI3L1; CHI3L2) were significantly increased while NOS2 and IL-1β mRNAs were unchanged.ConclusionImmune cells within the brain display gene profiles that suggest heterogeneous, functional phenotypes that range from a pro-inflammatory, classical activation state to an alternative activation state involved in repair and extracellular matrix remodeling. Our data suggest that innate immune cells in AD may exhibit a hybrid activation state that includes characteristics of classical and alternative activation.

Role of the different isoforms of cyclooxygenase and nitric oxide synthase during gastric ulcer healing in cyclooxygenase-1 and -2 knockout mice

Traditional NSAIDs, selective cyclooxygenase (COX)-2 inhibitors, and inhibitors of nitric oxide synthase (NOS) impair the healing of preexisting gastric ulcers. However, the role of COX-1 (with or without impairment of COX-2) and the interaction between COX and NOS isoforms during healing are less clear. Thus we investigated healing and regulation of COX and NOS isoforms during ulcer healing in COX-1 and COX-2 deficiency and inhibition mouse models. In this study, female wild-type COX-1(-/-) and COX-2(-/-) mice with gastric ulcers induced by cryoprobe were treated intragastrically with vehicle, selective COX-1 (SC-560), COX-2 (celecoxib, rofecoxib, and valdedoxib), and unselective COX (piroxicam) inhibitors. Ulcer healing parameters, mRNA expression, and activity of COX and NOS were quantified. Gene disruption or inhibition of COX-1 did not impair ulcer healing. In contrast, COX-2 gene disruption and COX-2 inhibitors moderately impaired wound healing. More severe healing impairment was found in dual (SC-560 + rofecoxib) and unselective (piroxicam) COX inhibition and combined COX impairment (in COX-1(-/-) mice with COX-2 inhibition and COX-2(-/-) mice with COX-1 inhibition). In the ulcerated repair tissue, COX-2 mRNA in COX-1(-/-) mice, COX-1 mRNA in COX-2(-/-) mice, and, remarkably, NOS-2 and NOS-3 mRNA in COX-impaired mice were more upregulated than in wild-type mice. This study demonstrates that COX-2 is a key mediator in gastric wound healing. In contrast, COX-1 has no significant role in healing when COX-2 is unimpaired but becomes important when COX-2 is impaired. As counterregulatory mechanisms, mRNA of COX and NOS isoforms were increased during healing in COX-impaired mice.

Neuronal Nitric Oxide Synthase Is Necessary for Elimination of Giardia lamblia Infections in Mice

NO produced by inducible NO synthase (NOS2) is important for the control of numerous infections. In vitro, NO inhibits replication and differentiation of the intestinal protozoan parasite Giardia lamblia. However, the role of NO against this parasite has not been tested in vivo. IL-6-deficient mice fail to control Giardia infections, and these mice have reduced levels of NOS2 mRNA in the small intestine after infection compared with wild-type mice. However, NOS2 gene-targeted mice and wild-type mice treated with the NOS2 inhibitor N-iminoethyl-L-lysine eliminated parasites as well as control mice. In contrast, neuronal NOS (NOS1)-deficient mice and wild-type mice treated with the nonspecific NOS inhibitor NG-nitro-L-arginine methyl ester and the NOS1-specific inhibitor 7-nitroindazole all had delayed parasite clearance. Finally, Giardia infection increased gastrointestinal motility in wild-type mice, but not in SCID mice. Furthermore, treatment of wild-type mice with NG-nitro-L-arginine methyl ester or loperamide prevented both the increased motility and the elimination of parasites. Together, these data show that NOS1, but not NOS2, is necessary for clearance of Giardia infection. They also suggest that increased gastrointestinal motility contributes to elimination of the parasite and may also contribute to parasite-induced diarrhea. Importantly, this is the first example of NOS1 being involved in the elimination of an infection.

Tissue Characterization of Hypoxia, Nitric Oxide and Apoptosis Pathways in a Baboon Model of E. coli Sepsis.

Sepsis - the most common cause of death in hospitalized patients -is characterized by severe inflammatory response and profound systemic vasculitic pathology accompanied by activation of blood coagulation. We observed previously that infusion of E. coli in baboons produced severe disruption of the microcirculation, such as capillary leakage, intravascular coagulation and tissue injury, which may lead to global tissue hypoxia, shock, organ failure, and death. Here, we report data on the temporal dynamics of tissue specific changes of hypoxia, nitric oxide (NO) and apoptosis pathways in response to sublethal or lethal E. coli challenge. Animals were administered sublethal doses of E. coli (LD30–50; 108 CFU/kg) and sacrificed after 2, 8 and 24 hours or lethal doses (LD100; 9x109 −1x1010 CFU/kg) for 8 hours. Both groups were compared to non challenged controls (n=3 per experimental condition). Six organs (lung, liver, heart, kidney, spleen and lymph nodes) were analyzed by immunofluorescence/confocal imaging for Hypoxia Inducible Factor 1 (HIF1α), NOS2, NOS3 nitrotyrosine (NT), inflammatory cell markers (myeloperoxidase and CD68 for neutrophils and macrophages, respectively), and apoptosis markers (active caspase-3, TUNEL, Bcl2, Bax). Tissue extracts were analyzed by ELISA (NT and caspase-3), dot and western blot (NT, Bcl2 and Bax) and RT-PCR and microarray assays for mRNA expression. The results show that HIF1α mRNA was increased by 2–3 fold, reaching maximum values at 2 hours in the lung and 24 hours in the liver. HIF1α nuclear translocation was detected by immunostaining at 8 hours in lung and 24 hours in the liver, especially in the LD100 E. coli challenged baboons. HIF1α functions as a master transcriptional regulator of the adaptive response to hypoxia, and controls the expression of over 40 genes including NOS2, an inducible enzyme that induces NO production and thus decreases the vascular tone, and VEGF, a very potent permeability factor. NOS2 mRNA and protein were induced after 8 hours mainly in inflammatory cells, while endothelial NOS3 showed a transient decrease at 2–8 hours, and increased at 24 hours as compared to controls. The overall increase in NO production was reflected in transient high levels of nitrosylated proteins at 8 hours in sublethal animals and persistent high levels in lethal baboons. Ultrastructural analysis of lung and kidney performed by electron microscopy showed signs of defective permeability, such as accumulation of plasma proteins in the lung alveolae and in the subendothelium of the kidney peritubular capillaries, concomitant with intra- and/or extravascular fibrin deposition. Dose-dependent induction of apoptosis was detected by TUNEL and caspase-3 staining, especially in the proximal contort tubes of the kidney, hepatocytes, lymphoid and pericapsular cells of spleen and lymph nodes. Mitochondrial manganese superoxide dismutase (SOD2), a protective enzyme produced in reaction to oxidative stress had a latter response, reaching the maximum mRNA expression at 8 hour in lung and 24 hours in the liver. Taken together, these results place hypoxia as a key factor in the development of organ dysfunction and multiple organ failure in sepsis and suggest that targeting hypoxia controlled transcription factors, such as HIF1α, or upregulating the antioxidant mechanisms may represent valuable therapeutic approaches.

Induction of Chronic Lymphocytic Leukemia (CLL) Apoptosis by Nitric Oxide Synthase (NOS) Inhibitors: Drug Efficacy Correlates with Lipid Solubility and NOS1 Dissociation Constant.

The viability of CLL cells may be dependent on the autocrine production of nitric oxide because nitric oxide synthase (NOS) inhibitors induce CLL cell apoptosis and CLL cells express inducible NOS (NOS2). Our previous study indicated that the non-specific NOS inhibitor NMMA induced CLL cell apoptosis but only at high concentrations (> 1 mM) (Levesque et al., Leukemia 17:442, 2003). Therefore, we performed the current study to identify NOS inhibitors that induce CLL cell apoptosis at lower concentrations and to understand factors that promote NOS inhibitor-induced CLL cell toxicity. We isolated and enriched CLL cells from the blood of CLL patients and cultured the CLL cells in media containing various concentrations of 21 different NOS inhibitors. We determined CLL cell viability following culture with each NOS inhibitor. We found that NOS inhibitors with specificity for neuronal NOS (NOS1) induced CLL cell death at concentrations lower than non-specific NOS inhibitors and lower than inducible NOS (NOS2) specific inhibitors. There was a weak correlation (r2 = 0.29, p = 0.1608) of the NOS1 (but not NOS2) half-maximal inhibitory concentration (IC50) of each NOS inhibitor for purified recombinant NOS and its ability to induce CLL cell death. We confirmed the specificity of the NOS inhibitors by inhibition of purified recombinant NOS1 and NOS2 enzyme activity, and we confirmed that NOS1 specific inhibitors induced CLL cell death by apoptosis. Because there was only a weak correlation of the NOS1 IC50 with NOS inhibitor induced CLL cell death, we considered whether other factors such as the Kd and hydrophobicity of each compound correlated with CLL cell death. We found that there was a direct correlation between the NOS1 (but not NOS2) dissociation constant (Kd) of NOS inhibitors and CLL cell death (r2 = 0.77, p = 0.0041) and a direct correlation of the partitioning coefficient (a measure of hydrophobicity) of each NOS inhibitor and its ability to induce CLL cell death (r2 = 0.68, p < 0.0001). Therefore, NOS inhibitors that bound tightly to NOS1 and were hydrophobic induced CLL cell death at lower concentrations. There was variable expression of CLL cell NOS1 mRNA (6 of 28 samples positive) and we were unable to demonstrate CLL cell expression of NOS1 protein by immunoblotting. This suggests that if NOS1 is present in CLL cells, it exists at very low levels. Taken together, we believe that low level NO production promotes CLL cell viability and that inhibition of CLL NOS induces CLL cell apoptosis. Importantly, our studies provide direction for the rational design and selection of NOS inhibitors that may be useful as CLL therapeutics.

Inhibition of interferon-γ signaling by a mercurio-substituted dihydropsoralen in murine keratinocytes

Psoralens and ultraviolet light A (PUVA) are used in the treatment of a variety of epidermal proliferative and inflammatory disorders. These compounds are known to intercalate and photo crosslink DNA. Specific receptor proteins for psoralens have also been identified. We describe a novel activity of a thiol reactive derivative, iodomercurio-4′,5′-dihydrotrimethylpsoralen (iodomercurio-H 2TMP) in keratinocytes. Without UVA, this psoralen was found to be an effective inhibitor of interferon-γ (IFN-γ)-signaling as measured by induction of nitric oxide biosynthesis (IC 50 = 0.8 μM). This activity was increased (IC 50 = 0.1 μM) when the cells were depleted of intracellular glutathione (GSH) with buthionine sulfoximine. In keratinocytes, IFN-γ stimulates expression of inducible nitric oxide synthase (NOS2). Although iodomercurio-H 2TMP did not alter NOS2 enzymatic activity, it blocked IFN-γ-induced expression of NOS2 mRNA and protein, an effect that was enhanced in GSH-depleted cells. Iodomercurio-H 2TMP was found to readily inhibit IFN-γ signaling in transient transfection assays using NOS2 promoter/luciferase reporter constructs. NOS2 gene expression is known to require a variety of transcription factors including STAT-1, NF-κB and AP-1. Using mobility shift assays the psoralen, at concentrations that inhibit nitric oxide biosynthesis, had no effect on the DNA binding activity of STAT-1 or NF-κB. However, iodomercurio-H 2TMP was found to suppress AP-1. These data indicate that iodomercurio-H 2TMP acts at sulfhydryl-sensitive sites to inhibit NOS2. Moreover, this is dependent on early events in the IFN-γ signal transduction pathway. Inhibition of AP-1 suggests that the psoralen functions by interfering with an important transcription factor that regulates expression of NOS2 in keratinocytes.

Weaning induces NOS-2 expression through NF-κB modulation in the lactating mammary gland: importance of GSH

At the end of lactation the mammary gland undergoes involution, a process characterized by apoptosis of secretory cells and tissue remodelling. To gain insight into this process, we analysed the gene expression profile by oligonucleotide microarrays during lactation and after forced weaning. Up-regulation of inflammatory mediators and acute-phase response genes during weaning was found. Expression of IkappaBalpha (inhibitory kappaBalpha), a protein known to modulate NF-kappaB (nuclear factor-kappaB) nuclear translocation, was significantly up-regulated. On the other hand, there was a time-dependent degradation of IkappaBalpha protein levels in response to weaning, suggesting a role for NF-kappaB. Furthermore, we have demonstrated, using chromatin immunoprecipitation assays, binding of NF-kappaB to the NOS-2 (inducible nitric oxide synthase) promoter at the early onset of events triggered during weaning. The three isoforms of NOS are constitutively present in the lactating mammary gland; however, while NOS-2 mRNA and protein levels and, consequently, NO production are increased during weaning, NOS-3 protein levels are diminished. Western blot analyses have demonstrated that protein nitration is increased in the mammary gland during weaning, but this is limited to a few specific tyrosine-nitrated proteins. Interestingly, inhibition of GSH synthesis at the peak of lactation partially mimics these findings, highlighting the role of NO production and GSH depletion during involution.