Inducible Nitric Oxide Synthase Knockout Mice Research Articles

A Critical Role for IFN Regulatory Factor 1 in NKT Cell-Mediated Liver Injury Induced by α-Galactosylceramide

NKT cells are remarkably abundant in mouse liver. Compelling experimental evidence has suggested that NKT cells are involved in the pathogenesis of many liver diseases. Activation of NKT cells with alpha-galactosylceramide (alpha-GalCer) causes liver injury through mechanisms that are not well understood. We undertook studies to characterize the key pathways involved in alpha-GalCer-induced liver injury. We found that expression of the transcription factor IFN regulatory factor 1 (IRF-1) in mouse liver was dramatically upregulated by alpha-GalCer treatment. Neutralization of either TNF-alpha or IFN-gamma inhibited alpha-GalCer-mediated IRF-1 upregulation. alpha-GalCer-induced liver injury was significantly suppressed in IRF-1 knockout mice or in wild-type C56BL/6 mice that received a microRNA specifically targeting IRF-1. In contrast, overexpression of IRF-1 greatly potentiated alpha-GalCer-induced liver injury. alpha-GalCer injection also induced a marked increase in hepatic inducible NO synthase expression in C56BL/6 mice, but not in IRF-1 knockout mice. Inducible NO synthase knockout mice exhibited significantly reduced liver injury following alpha-GalCer treatment. Finally, we demonstrated that both NKT cells and hepatocytes expressed IRF-1 in response to alpha-GalCer. However, it appeared that the hepatocyte-derived IRF-1 was mainly responsible for alpha-GalCer-induced liver injury, based on the observation that inhibition of IRF-1 by RNA interference did not affect alpha-GalCer-induced NKT cell activation. Our findings revealed a novel mechanism of NKT cell-mediated liver injury in mice, which has implications in the development of human liver diseases.

Myelin basic protein priming reduces the expression of Foxp3 in T cells via nitric oxide.

Regulatory T cells (Tregs) play a vital role in autoimmune disorders. Among several markers, forkhead box p3 (Foxp3) is the most specific with regard to Treg activity. Therefore, understanding mechanisms that regulate Foxp3 expression is a critical step for unraveling the complicacy of autoimmune pathophysiology. The present study was undertaken to investigate the crosstalk between NO and Tregs. Interestingly, after myelin basic protein (MBP) priming, the expression of Foxp3 decreased in MBP-primed T cells. However, blocking NO either by inhibiting inducible NO synthase with l-N(6)-(1-iminoethyl)-lysine hydrochloride or through scavenging with PTIO or by pharmacological drugs, such as pravastatin, sodium benzoate, or gemfibrozil, restored the expression of Foxp3 in MBP-primed T cells. However, this restoration of Foxp3 by pharmacological drugs was reversed by S-nitrosoglutathione, an NO donor. Similarly, NO also decreased the populations of Tregs characterized by CD4(+)CD25(+) and CD25(+)FoxP3(+) phenotypes. We have further confirmed this inverse relationship between NO and Foxp3 by analyzing the mRNA expression of Foxp3 and characterizing CD25(+)FoxP3(+) or CD4(+)Foxp3(+) phenotypes from inducible NO synthase knockout mice. Moreover, this inverse relation between NO and Foxp3 also was observed during priming with myelin oligodendrocyte glycoprotein, another target neuroantigen in multiple sclerosis, as well as collagen, a target autoantigen in rheumatoid arthritis. Finally, we demonstrate that NO inhibited the expression of Foxp3 in MBP-primed T cells via soluble guanylyl cyclase-mediated production of cGMP. Taken together, our data imply a novel role of NO in suppressing Foxp3(+) Tregs via the soluble guanylyl cyclase pathway.

IFN-γ Acts Directly on Activated CD4+ T Cells during Mycobacterial Infection to Promote Apoptosis by Inducing Components of the Intracellular Apoptosis Machinery and by Inducing Extracellular Proapoptotic Signals

Despite many studies, the regulation of CD4(+) T cell apoptosis during the shutdown of immune responses is not fully understood. We have investigated the molecular mechanisms of IFN-gamma in regulating apoptosis of CD4(+) T cells during bacillus Calmette-Guérin (BCG) infection of mice. Our data provide new insight into the regulation of CD4(+) T cell apoptosis by IFN-gamma. As CD4(+) T cells responded to BCG infection, there was a coordinated increase in IFN-gamma production by effector CD4(+) T cells and a coordinated IFN-gamma-dependent up-regulation of many diverse apoptosis-pathway genes in effector CD4(+) T cells. Unexpectedly, IFN-gamma up-regulated transcripts and protein expression of Bcl-2, Bax, Bim, Bid, Apaf-1, and caspase-9 in activated CD4(+) T cells--components of the apoptosis machinery that are involved in promoting mitochondrial damage-mediated apoptosis. Wild-type, but not IFN-gamma knockout, CD4(+) T cells underwent apoptosis that was associated with damaged mitochondrial membranes. IFN-gamma also up-regulated expression of cell-extrinsic signals of apoptosis, including TRAIL, DR5, and TNFR1. Cell-extrinsic apoptosis signals from TNF-alpha, TRAIL, and NO were capable of damaging the mitochondrial membranes in activated CD4(+) T cells. Moreover, activated CD4(+) T cells from BCG-infected DR5, TNFR1, and inducible NO synthase knockout mice had impaired caspase-9 activity, suggesting impaired mitochondria-pathway apoptosis. We propose that IFN-gamma promotes apoptosis of CD4(+) T cells during BCG infection as follows: 1) by sensitizing CD4(+) T cells to apoptosis by inducing intracellular apoptosis molecules and 2) by inducing cell-extrinsic apoptosis signals that kill CD4(+) effector T cells.

Nitric oxide inhibits enterocyte migration through activation of RhoA-GTPase in a SHP-2-dependent manner

Diseases of intestinal inflammation like necrotizing enterocolitis (NEC) are associated with impaired epithelial barrier integrity and the sustained release of intestinal nitric oxide (NO). NO modifies the cytoskeletal regulator RhoA-GTPase, suggesting that NO could affect barrier healing by inhibiting intestinal restitution. We now hypothesize that NO inhibits enterocyte migration through RhoA-GTPase and sought to determine the pathways involved. The induction of NEC was associated with increased enterocyte NO release and impaired migration of bromodeoxyuridine-labeled enterocytes from terminal ileal crypts to villus tips. In IEC-6 enterocytes, NO significantly inhibited enterocyte migration and activated RhoA-GTPase while increasing the formation of stress fibers. In parallel, exposure of IEC-6 cells to NO increased the phosphorylation of focal adhesion kinase (pFAK) and caused a striking increase in cell-matrix adhesiveness, suggesting a mechanism by which NO could impair enterocyte migration. NEC was associated with increased expression of pFAK in the terminal ileal mucosa of wild-type mice and a corresponding increase in disease severity compared with inducible NO synthase knockout mice, confirming the dependence of NO for FAK phosphorylation in vivo and its role in the pathogenesis of NEC. Strikingly, inhibition of the protein tyrosine phosphatase SHP-2 in IEC-6 cells prevented the activation of RhoA by NO, restored focal adhesions, and reversed the inhibitory effects of NO on enterocyte migration. These data indicate that NO impairs mucosal healing by inhibiting enterocyte migration through activation of RhoA in a SHP-2-dependent manner and support a possible role for SHP-2 as a therapeutic target in diseases of intestinal inflammation like NEC.

Mesenchymal Stem Cells Produce Nitric Oxide, a Key Molecule for T Cell Suppression, upon Interaction with Activated T Cells.

The molecular mechanisms by which mesenchymal stem cells (MSCs) suppress T cell proliferation are poorly understood, and whether a soluble factor plays a major role remains controversial. We demonstrated that nitric oxide (NO) is important for T cell suppression by MSCs (ASH 2005, 403a). Here we further demonstrate that the production of NO in the presence of MSCs is dependent on CD4 or CD8 T cells but not on CD19 B cells. MSCs inhibits B cell proliferation induced by LPS, suggesting that mechanisms of supppression by MSCs are different between T cells and B cells. Inducible NO synthase was exclusively detected in MSCs co-cultured with activated T cells, indicating that the producer of NO is MSC. Experiments with transwell system revealed that separation by transwell membrane reduces the induction of NO and T cell suppression. RAW246.7 macrophage cell line showed a similar transwell-mediated inhibition, suggesting that the inhibition by transwell is a common feature of NO and that direct contact is critical for efficient NO production and T cell suppression. Furthermore, inhibitors of prostaglandin synthase or NO synthase restored the proliferation of T cells, whereas an inhibitor of indoleamine 2,3-dioxygenase and a transforming growth factor-β-neutralizing antibody had no effect. In the view of that NO is upstream of PGE2, NO may be a key regulator of T cell suppression induced by MSCs. Finally, we used inducible NO synthase knockout mice to reconfirm all results here. MSCs from knockout mice did not produce NO even in the presence of activated wild type T cells and had a reduced ability to suppress T cell proliferation. Meanwhile, proliferation of splenocytes from knockout mice was suppressed in the presence of wild type MSCs and NO production was readily detected, confirming that NO produced by MSCs plays a critical role in T cell suppression.

A novel role for suppressor of cytokine signaling 3 in cartilage destruction via induction of chondrocyte desensitization toward insulin‐like growth factor

An important mechanism contributing to cartilage destruction in arthritis is chondrocyte desensitization toward its main anabolic factor, insulin-like growth factor 1 (IGF-1). In this study, we sought to determine the role of suppressor of cytokine signaling 3 (SOCS-3) in the induction of IGF-1 desensitization of murine chondrocytes. Chondrocyte responsiveness to IGF-1 was assessed by 35S-sulfate incorporation into proteoglycans (PGs), via aggrecan messenger RNA expression, using quantitative real-time polymerase chain reaction or insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation (Western blot analysis). IGF-1 desensitization of patellar chondrocytes was studied in zymosan-induced arthritis. IGF-1 desensitization was induced in patellar cartilage explants or the H4 chondrocyte cell line, exposed to interleukin-1alpha (IL-1alpha). SOCS-3 protein expression was assessed by immunohistochemistry or by Western blot analysis of protein extracts. The role of SOCS-3 in IGF-1 signaling was elucidated by adenoviral overexpression. Exposure of murine articular cartilage to IL-1 caused a significant decrease in IGF-1-induced PG synthesis. This effect also occurred in inducible nitric oxide synthase-knockout mice, revealing the involvement of a secondary IL-1-induced factor other than nitric oxide. We showed that IL-1 significantly up-regulated SOCS-3 transcription and protein synthesis in H4 chondrocytes. In contrast, IL-18 was unable to induce SOCS-3 expression and failed to induce chondrocyte IGF-1 desensitization. Histologic analysis of samples from arthritic knee joints revealed high expression of SOCS-3 in chondrocytes. Through adenoviral overexpression of SOCS-3, we obtained direct evidence that SOCS-3 inhibits IGF-1-mediated cell signaling, since IRS-1 phosphorylation was reduced. This study demonstrates that IL-1-induced SOCS-3 expression is a novel mechanism of IGF-1 desensitization in chondrocytes; in conjunction with nitric oxide it can contribute to cartilage damage during arthritis.

Interaction between enteric epithelial cells and Peyer’s patch lymphocytes in response to Shigella lipopolysaccharide: Effect on nitric oxide and IL-6 release

To investigate the effect of interaction between enteric epithelial cells and lymphocytes of Peyer's patch on the release of nitric oxide (NO) and IL-6 in response to Shigella lipopolysaccharide (LPS). Human colonic epithelial cells (Caco-2) were mixed cocultured with lymphocytes of Peyer's patch from wild-type (C57 mice) and inducible NO synthase knockout mice, and challenged with Shigella F2a-12 LPS. Release of NO and mIL-6 was measured by Griess colorimetric assay and enzyme-linked immunosorbent assay (ELISA), respectively. In the absence of LPS challenge, NO was detected in the culture medium of Caco-2 epithelial cells but not in lymphocytes of Peyer's patch, and the NO release was further up-regulated in both cocultures with lymphocytes from either the wild-type or iNOS knockout mice, with a significantly higher level observed in the coculture with iNOS knockout lymphocytes. After Shigella F2a-12 LPS challenge for 24-h, NO production was significantly increased in both Caco-2 alone and the coculture with lymphocytes of Peyer's patch from the wild-type mice but not from iNOS knockout mice. LPS was found to stimulate the release of mIL-6 from lymphocytes, which was suppressed by coculture with Caco-2 epithelial cells. The LPS-induced mIL-6 production in lymphocytes from iNOS knockout mice was significantly greater than that from the wild-type mice. Lymphocytes of Peyer's patch maintain a constitutive basal level of NO production from the enteric epithelial cell Caco-2. LPS-induced mIL-6 release from lymphocytes of Peyer's patch is suppressed by the cocultured epithelial cells. While no changes are detectable in NO production in lymphocytes from both wild-type and iNOS knockout mice before and after LPS challenge, NO from lymphocytes appears to play an inhibitory role in epithelial NO release and their own mIL-6 release in response to LPS.

Astroglia induce cytotoxic effects on brain tumors via a nitric oxide-dependent pathway both in vitro and in vivo.

In the central nervous system, astroglia produce nitric oxide (NO) in response to cytokines. We investigated whether cytokine stimulation of astroglia could inhibit brain tumor cell growth in vitro and prolong survival in vivo via an NO-dependent pathway. Astroglia cultures were stimulated with the cytokines lipopolysaccharide and interferon-gamma and subsequently seeded with tumor cell lines. Wild-type mice and inducible NO synthase-knockout mice received in vivo cytokine stimulation followed by B16F10 murine melanoma challenge. Our in vitro studies demonstrate that astroglia stimulated to produce NO by the addition of cytokines dose-dependently inhibit the growth of one primary rat brain tumor cell line (9L) and three primary human brain tumor cell lines (H80, U87, and U373). This inhibition of tumor cell growth is also observed in metastatic cell lines (B16F10 melanoma, Lewis lung carcinoma, and CT26 colon). Cultured astrocytes from inducible NO synthase-knockout mice, which are incapable of induction of NO, are without the enhanced tumoricidal effect. Furthermore, when C57BL/6 mice are primed to produce NO through stereotactic intracranial administration of lipopolysaccharide plus interferon-gamma and subsequently challenged with B16F10 murine melanoma, survival is significantly prolonged, with a median survival of 26 days versus 16 days in the control group (P < 0.001). The addition of an NO synthase inhibitor (N(G)-nitro-l-arginine methyl ester) decreases this beneficial effect (median survival, 21 d). These findings suggest that NO may have an important role as a defense mechanism molecule against brain tumors; stimulation or modification of this mechanism may represent a new approach to the treatment of primary and metastatic brain tumors.

Regulation of vascular endothelial growth factor gene expression in murine macrophages by nitric oxide and hypoxia.

Vascular endothelial growth factor (VEGF) expression in murine peritoneal macrophages is strongly upregulated by hypoxia via transcriptional and posttranscriptional mechanisms. Interferon-gamma (IFN-gamma) with Escherichia coli lipopolysaccharide (LPS) also upregulates expression of VEGF, as well as of the inducible nitric oxide synthase (iNOS). Hypoxia (1% O(2)) upregulates VEGF expression in macrophages from both wild-type and iNOS knockout mice, indicating that hypoxic upregulation of VEGF is independent of iNOS. However, the iNOS inhibitor aminoguanidine (AG) decreases the VEGF expression induced by LPS/IFN-gamma, indicating an important role for NO. NO-dependent induction of VEGF is strongly dependent on cell density. LPS/IFN-gamma treatment induces minimal VEGF protein expression in macrophages cultured at low cell densities (<0.25 x 10(6) cells/cm(2)); at higher cell densities (>0.25 x 10(6) cells/cm(2)) that lead to conditions of pericellular hypoxia, however, induction of VEGF expression was strong. Transient transfection of RAW 264.7 cells with luciferase reporter constructs of the murine VEGF promoter indicates that both hypoxia and LPS/IFN-gamma independently induce VEGF promoter activity, irrespective of cell density. Although LPS/IFN-gamma treatment induces transcriptional activation of the VEGF promoter, significant levels of VEGF protein are only expressed by cells at high density under conditions of pericellular hypoxia. This suggests an important regulatory role for hypoxia at the posttranscriptional level. Deletion analysis of the VEGF promoter shows that the hypoxia response element region and its immediate flanking sequences are essential for both hypoxia and LPS/IFN-gamma-induced VEGF promoter activation.

Nitric oxide partially controls Coxiella burnetii phase II infection in mouse primary macrophages.

In most primary or continuous cell cultures infected with the Q-fever agent Coxiella burnetii, bacteria are typically sheltered in phagolysosome-like, large replicative vacuoles (LRVs). We recently reported that only a small proportion of mouse peritoneal macrophages (PMPhi) infected with a nonvirulent, phase II strain of C. burnetii developed LRVs and that their relative bacterial load increased only slowly. In the majority of infected PMPhi, the bacteria were confined to the small vesicles. We show here that nitric oxide (NO) induced by the bacteria partially accounts for the restricted development of LRVs in primary macrophages. Thus, (i) PMPhi and bone marrow-derived macrophages (BMMPhi) challenged with phase II C. burnetii produced significant amounts of NO; (ii) the NO synthase inhibitors aminoguanidine and N-methyl-L-arginine reduced the production of NO and increased the frequency of LRVs (although the relative bacterial loads of individual LRVs did not change, the estimated loads per well increased appreciably); (iii) gamma interferon (IFN-gamma) or the NO donor sodium nitroprusside, added to BMMPhi prior to or after infection, reduced the development and the relative bacterial loads of LRVs and lowered the yield of viable bacteria recovered from the cultures; and (iv) these effects of IFN-gamma may not be entirely dependent on the production of NO since IFN-gamma also controlled the infection in macrophages from inducible NO synthase knockout mice. It remains to be determined whether NO reduced the development of LRVs by acting directly on the bacteria; by acting on the traffic, fusion, or fission of cell vesicles; or by a combination of these mechanisms.

Myeloid suppressor lines inhibit T cell responses by an NO-dependent mechanism.

CD11b(+)Gr-1(+) myeloid suppressor cells (MSC) accumulate in lymphoid organs under conditions of intense immune stress where they inhibit T and B cell function. We recently described the generation of immortalized MSC lines that provide a homogeneous source of suppressor cells for dissecting the mechanism of suppression. In this study we show that the MSC lines potently block in vitro proliferation of T cells stimulated with either mitogen or antigenic peptide, with as few as 3% of MSC cells causing complete suppression. Inhibition of mitogenic and peptide-specific responses is not associated with a loss in IL-2 production or inability to up-modulate the early activation markers, CD69 and CD25, but results in direct impairment of the three IL-2R signaling pathways, as demonstrated by the lack of Janus kinase 3, STAT5, extracellular signal-regulated kinase, and Akt phosphorylation in response to IL-2. Suppression is mediated by and requires NO, which is secreted by MSC in response to signals from activated T cells, including IFN-gamma and a contact-dependent stimulus. Experiments with inducible NO synthase knockout mice demonstrated that the inhibition of T cell proliferation by CD11b(+)Gr-1(+) cells in the spleens of immunosuppressed mice is also dependent upon NO, indicating that the MSC lines accurately represent their normal counterparts. The distinctive capacity of MSC to generate suppressive signals when encountering activated T cells defines a specialized subset of myeloid cells that most likely serve a regulatory function during times of heightened immune activity.

Inducible Nitric Oxide Synthase—Knockout Mice Exhibit Resistance to Pleurisy and Lung Injury Caused by Carrageenan

In the present study, we investigated the role of inducible (or type 2) nitric oxide synthase (iNOS) in the development of acute inflammation by comparing the responses in wild-type mice (WT) and mice lacking (knockout [KO]). When compared with carrageenan-treated iNOS-WT mice, iNOS-KO mice that had received carrageenan exhibited a reduced degree of pleural exudation and polymorphonuclear cell migration. Lung myeloperoxidase (MPO) activity and lipid peroxidation were significantly reduced in iNOS-KO mice in comparison with iNOSWT mice. Immunohistochemical analysis for nitrotyrosine revealed positive staining in lungs from carrageenan-treated iNOS-WT mice. Lung tissue sections from carrageenan-treated iNOS-WT mice showed positive staining for poly adenosine diphosphate (ADP)-ribose synthetase that was mainly localized in alveolar macrophages and in airway epithelial cells. The intensity and degree of staining for nitrotyrosine and poly-ADP-ribose synthetase were markedly reduced in tissue sections from carrageenan-treated iNOS-KO mice. The inflamed lungs of iNOS-KO mice also showed an improved histologic status. Furthermore, a significant reduction in the suppression of energy status, in DNA strand breakage, and in decreased cellular levels of nicotinamide adenine dinucleotide (NAD(+)) was observed ex vivo in macrophages harvested from the pleural cavity of iNOS-KO mice subjected to carrageenan-induced pleurisy. Taken together, our results clearly show that iNOS plays an important role in the acute inflammatory response.