Is inducible nitric oxide synthase a target for chemoprevention?

The molecular messenger nitric oxide (NO) is synthesized endogenously from L-arginine by three isoforms of the enzyme NO synthase. The isoform most consistently associated with neoplasia is the inducible form, inducible nitric oxide synthase (iNOS). However, the role played by the NO/iNOS system in tumor development is complex, and both promoting and inhibitory effects on neoplasia have been reported. This review attempts to clarify the role of iNOS in carcinogenesis, with particular emphasis on the early stages of tumor development, offers possible explanations for the confused picture presented in the literature regarding the association of the NO/iNOS pathway with neoplasia, and identifies selective iNOS inhibitors that may have chemopreventive potential.     

Does upregulation of inducible nitric oxide synthase play a role in hepatic injury?

Lipopolysaccharide (LPS) and gut ischemia/reperfusion (I/R) injury cause reversible liver injury. Because nitric oxide (NO) can have both beneficial and deleterious effects in the gastrointestinal tract, and because the role of NO in gut I/R-induced hepatic injury is unknown, this study examined its role in LPS and gut I/R-induced hepatic injury in the rat. Both LPS and gut I/R caused a similar increase in serum hepatocellular enzymes. LPS but not gut I/R caused a significant increase in upregulation of hepatic inducible NO synthase (iNOS) according to quantitative real-time RT-PCR and Western immunoblot analysis. Aminoguanidine, a selective iNOS inhibitor, attenuated LPS-induced hepatic injury and hypotension, but did not prevent gut I/R-induced hepatic injury. In contrast, the non-selective NOS inhibitor N(G)-nitro-L-arginine methyl ester aggravated liver damage from both LPS and gut I/R. These data indicate that iNOS plays a role in mediating LPS-induced hepatic injury, but not gut I/R-induced hepatic injury. The data also suggest that the constitutive isoforms of NOS play a hepatoprotective role in both models of hepatic injury.     

Physiologic role for“inducible”nitric oxide synthase:A new form of astrocytic-neuronal interface

长期以来,一氧化氮(NO)都被看作是影响兴奋性突触传递的非典型神经信使分子,其细胞来源尚不清楚.许多脑区中的神经元型一氧化氮合酶(nNOS)只在少量抑制性神经元中表达.众所周知,胶质细胞诱导型一氧化氮合酶(iNOS)不在正常大脑中表达,其在免疫刺激后可经转录介导上调.因此,iNOS调节正常神经功能的作用常被忽视.许多研...     

Inducible nitric oxide synthase and nitric oxide production inLeishmania infantum-infected human macrophages stimulated with interferon-γ and bacterial lipopolysaccharide

Nitric oxide produced by an inducible nitric oxide synthase constitutes one of the main microbicidal mechanisms of murine macrophages and its importance is now being recognized for human macrophages. In this study we evaluated inducible nitric oxide synthase expression, nitric oxide release, and parasitocidal ability of Leishmania infantum-infected monocyte-derived human macrophages. The inducible nitric oxide synthase was detected by immunofluorescence and western blotting and nitric oxide production was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Experiments were performed on macrophages with or without previous stimulation with recombinant human interferon-gamma and bacterial lipopolysaccharide. Inducible nitric oxide synthase expression and nitric oxide release were higher in Leishmania-infected stimulated macrophages than in uninfected cells or infected cells without previous stimulation. Nitric oxide production and parasitocidal activity against Leishmania infantum were reduced in macrophages treated with the nitric oxide synthase inhibitor L-N(G) monomethylarginine. These results suggest a microbicidal role for nitric oxide in human leishmaniasis, with the possible practical application of immunological or pharmacological regulation of nitric oxide synthesis in the treatment of this infection.     

Memory T cell–driven differentiation of naive cells impairs adoptive immunotherapy

Adoptive cell transfer (ACT) of purified naive, stem cell memory, and central memory T cell subsets results in superior persistence and antitumor immunity compared with ACT of populations containing more-differentiated effector memory and effector T cells. Despite a clear advantage of the less-differentiated populations, the majority of ACT trials utilize unfractionated T cell subsets. Here, we have challenged the notion that the mere presence of less-differentiated T cells in starting populations used to generate therapeutic T cells is sufficient to convey their desirable attributes. Using both mouse and human cells, we identified a T cell–T cell interaction whereby antigen-experienced subsets directly promote the phenotypic, functional, and metabolic differentiation of naive T cells. This process led to the loss of less-differentiated T cell subsets and resulted in impaired cellular persistence and tumor regression in mouse models following ACT. The T memory–induced conversion of naive T cells was mediated by a nonapoptotic Fas signal, resulting in Akt-driven cellular differentiation. Thus, induction of Fas signaling enhanced T cell differentiation and impaired antitumor immunity, while Fas signaling blockade preserved the antitumor efficacy of naive cells within mixed populations. These findings reveal that T cell subsets can synchronize their differentiation state in a process similar to quorum sensing in unicellular organisms and suggest that disruption of this quorum-like behavior among T cells has potential to enhance T cell–based immunotherapies.     

Preventive non–pharmacological treatment and nitric oxide in chronic migraine

In chronic migraine the central sensitisation and the changes of regional cerebral blood flow are mediated by nitric oxide (NO) and oxygen free radicals. Biofeedback is considered a preventive non–pharmacological treatment decreasing migraine attacks. We investigated whether biofeedback effectiveness is related to relaxation processes and its influence on oxidative stress. The Migraine Disability Assessment Score (MIDAS) and serum NO stable metabolites (NO_x) were evaluated in 20 patients with chronic migraine before and at the end of biofeedback sessions. MIDAS score was lower after biofeedback than that reported before treatment. NO_x serum levels were higher after biofeedback than those measured before starting treatment. Thus, the effectiveness of biofeedback is related to a muscular relaxation and to its influence on NO bioavailability in patients with chronic migraine.     

Hepatocellular damage in porcine endotoxemia: beneficial effects of selective versus non-selective nitric oxide synthase inhibition?

While nitric oxide (NO) is implicated as an important mediator of hypotension in sepsis and endotoxemia, its role as a mediator of tissue injury in shock is controversial. During porcine endotoxemia (lipopolysaccharide (LPS) 1.7 microg kg(-1) x h(-1) i.v. for 6 h), we compared circulatory and morphological changes in the liver induced by two different NO synthase inhibitors (N(G)-nitro-L-arginine methyl ester, L-NAME, 25 mg x kg(-1) i.v. and aminoethyl-isothiourea, AE-ITU, 10 mg x kg(-1) i.v.), both given after 3 h. LPS induced time-dependent tissue reactions with edema, sinusoidal dilation, packing of red cells and leukocyte infiltration, progressing to endothelial cell and hepatocyte damage, formation of thrombi, and at 6 h widespread necrosis. These changes were similar in all pigs receiving LPS, regardless of treatment with NOS inhibitors. LPS caused significant increases in aspartate aminotransferase (AST), alkaline phosphatase (ALP) and alpha glutathione S-transferase (alpha-GST), L-NAME caused further increases in AST, ALP and alpha-GST, while AE-ITU prevented the late increase in ALP and alpha-GST observed in the other LPS groups. LPS reduced liver blood flow by approximately 40%. L-NAME further reduced flow by approximately 50%, while AE-ITU restored liver blood flow to baseline values. CONCLUSION: L-NAME in endotoxemia had detrimental effects on liver circulation, while AE-ITU improved liver blood flow and attenuated the late increase in liver enzymes. Liver morphology was unaffected within the 3-h observation time after NOS inhibition.     

BM mesenchymal stromal cell–derived exosomes facilitate multiple myeloma progression

BM mesenchymal stromal cells (BM-MSCs) support multiple myeloma (MM) cell growth, but little is known about the putative mechanisms by which the BM microenvironment plays an oncogenic role in this disease. Cell-cell communication is mediated by exosomes. In this study, we showed that MM BM-MSCs release exosomes that are transferred to MM cells, thereby resulting in modulation of tumor growth in vivo. Exosomal microRNA (miR) content differed between MM and normal BM-MSCs, with a lower content of the tumor suppressor miR-15a. In addition, MM BM-MSC–derived exosomes had higher levels of oncogenic proteins, cytokines, and adhesion molecules compared with exosomes from the cells of origin. Importantly, whereas MM BM-MSC–derived exosomes promoted MM tumor growth, normal BM-MSC exosomes inhibited the growth of MM cells. In summary, these in vitro and in vivo studies demonstrated that exosome transfer from BM-MSCs to clonal plasma cells represents a previously undescribed and unique mechanism that highlights the contribution of BM-MSCs to MM disease progression.     

T cell–derived interleukin (IL)-21 promotes brain injury following stroke in mice

T lymphocytes are key contributors to the acute phase of cerebral ischemia reperfusion injury, but the relevant T cell–derived mediators of tissue injury remain unknown. Using a mouse model of transient focal brain ischemia, we report that IL-21 is highly up-regulated in the injured mouse brain after cerebral ischemia. IL-21–deficient mice have smaller infarcts, improved neurological function, and reduced lymphocyte accumulation in the brain within 24 h of reperfusion. Intracellular cytokine staining and adoptive transfer experiments revealed that brain-infiltrating CD4⁺ T cells are the predominant IL-21 source. Mice treated with decoy IL-21 receptor Fc fusion protein are protected from reperfusion injury. In postmortem human brain tissue, IL-21 localized to perivascular CD4⁺ T cells in the area surrounding acute stroke lesions, suggesting that IL-21–mediated brain injury may be relevant to human stroke.Stroke is one of the leading causes of death and disability worldwide. Clinical and preclinical experimental studies highlight the importance of inflammation in both acute and delayed neuronal tissue damage after ischemic stroke; however, the mechanisms and cells involved in this neuroinflammation are not fully understood. There is currently no available treatment targeting the acute immune response that develops in the brain after transient focal ischemia. Therefore, we sought to identify novel T cell–derived cytokines that contribute to acute cerebral reperfusion using the mouse model of transient middle cerebral artery occlusion (tMCAO).During the reperfusion of infarcted brain tissue, leukocytes accumulate in the injured brain where, in addition to clearing cell debris, they promote secondary tissue injury (Yilmaz and Granger, 2010). Within the acute phase of ischemic reperfusion (I/R) injury there are multiple waves of cell infiltration of macrophages, neutrophils, and lymphocytes (Gelderblom et al., 2009). Brain-infiltrating T cells have also been widely reported in stroke and animal models of stroke and are thought to have acute detrimental and delayed protective effects (Magnus et al., 2012). Conventionally, the protective role of T cells has been attributed to the accumulation of regulatory T cells within the CNS in later stages of reperfusion injury. These T cells produce a variety of cytokines including TGFβ and IL-10, which are both antiinflammatory and neuroprotective. (Liesz et al., 2009; Stubbe et al., 2013). In addition to having an established role in delayed neuroprotection, Kleinschnitz et al. (2013) have recently shown that CD4⁺ CD25⁺ regulatory T cells also promote acute ischemic injury through interaction with the cerebral vasculature. The acute detrimental effects can be further divided into early (24 h) and late (72 h) phases, with IL-17 production by nonconventional γδ T cells (less common T cell subset associated with mucosal tissues) possibly accounting for the latter by promoting neutrophil accumulation (Gelderblom et al., 2012).The mechanisms of the early detrimental effects of T cells after cerebral ischemia are least understood. Several laboratories have reported reduced neurological deficit and infarct volumes at 24–48 h reperfusion in T cell–deficient mice after tMCAO (Yilmaz and Granger, 2010). After tMCAO, recombination activating gene 1–deficient (RAG1 KO) mice, which lack T and B lymphocytes, have significantly smaller brain injury compared with controls; whereas, adoptive transfer of WT CD4⁺ helper T cells or CD8⁺ cytotoxic T cells increases stroke infarct volumes within 24 h after ischemia in these mice (Kleinschnitz et al., 2010). Additionally, TCR-transgenic mice and mice lacking co-stimulatory TCR signaling molecules were fully susceptible to acute I/R injury, indicating that T cell involvement at early time points is antigen-independent (Kleinschnitz et al., 2010). These data demonstrate that conventional CD4⁺ or CD8⁺ αβ T cells exacerbate acute injury after cerebral ischemia independently of TCR ligation, and this effect seems to be concomitant with an early increase in T cell infiltration into the postischemic brain, which many have reported to be between 3 and 48 h (Yilmaz et al., 2006; Gelderblom et al., 2009).Recent findings suggest that, in the postischemic brain, within hours of reperfusion T cells accumulate in postcapillary segments of periinfarct inflamed cerebral microvasculature characterized by high endothelial expression of chemokines and adhesion molecules. These postcapillary venules have been postulated to allow accumulating immune cells to activate each other and promote platelet adhesion in a process termed thrombo-inflammation (Nieswandt et al., 2011). Much research has been devoted to identifying T cell factors that promote thrombo-inflammation (Barone et al., 1997; Hedtjärn et al., 2002; Yilmaz et al., 2006; Shichita et al., 2009; Gelderblom et al., 2012); however, to our knowledge no study has yet identified the T cell–derived factors responsible for the early increase in infarct volumes at 24 h reperfusion. Here, we present data that identify IL-21 as a key CD4⁺ T cell–derived inflammatory factor that contributes to increased early ischemic tissue injury.     

Immune cell–derived opioids protect against neuropathic pain in mice

The analgesic effects of leukocyte-derived opioids have been exclusively demonstrated for somatic inflammatory pain, for example, the pain associated with surgery and arthritis. Neuropathic pain results from injury to nerves, is often resistant to current treatments, and can seriously impair a patient’s quality of life. Although it has been recognized that neuronal damage can involve inflammation, it is generally assumed that immune cells act predominately as generators of neuropathic pain. However, in this study we have demonstrated that leukocytes containing opioids are essential regulators of pain in a mouse model of neuropathy. About 30%–40% of immune cells that accumulated at injured nerves expressed opioid peptides such as β-endorphin, Met-enkephalin, and dynorphin A. Selective stimulation of these cells by local application of corticotropin-releasing factor led to opioid peptide–mediated activation of opioid receptors in damaged nerves. This ultimately abolished tactile allodynia, a highly debilitating heightened response to normally innocuous mechanical stimuli, which is symptomatic of neuropathy. Our findings suggest that selective targeting of opioid-containing immune cells promotes endogenous pain control and offers novel opportunities for management of painful neuropathies.     

Mast cell–derived particles deliver peripheral signals to remote lymph nodes

During infection, signals from the periphery are known to reach draining lymph nodes (DLNs), but how these molecules, such as inflammatory cytokines, traverse the significant distances involved without dilution or degradation remains unclear. We show that peripheral mast cells, upon activation, release stable submicrometer heparin-based particles containing tumor necrosis factor and other proteins. These complexes enter lymphatic vessels and rapidly traffic to the DLNs. This physiological drug delivery system facilitates communication between peripheral sites of inflammation and remote secondary lymphoid tissues.The draining LNs (DLNs) are dynamic lymphoid structures that coordinate the development of specific immune responses after microbial or vaccine challenge. In response to these peripheral events, the DLN quickly undergoes significant structural changes, including rapid growth and vascular remodeling (McLachlan et al., 2003; Soderberg et al., 2005). This enlargement, which is largely attributable to enhanced recruitment and retention of naive lymphocytes from the circulation, increases the probability that rare lymphocytes bearing relevant specificities will be present to interact with activated tissue-derived APCs, which must migrate from inflamed tissues via afferent lymphatic vessels. This interaction between lymphocytes and APCs occurring within DLNs is the critical initiating event in the development of the adaptive immune response.For these rapid and impressive changes in DLNs to occur after microbial or vaccine challenge, not only should lymphocytes and APCs be able to reach the DLN but, importantly, LNs must receive signals from the periphery to create the appropriate environment to facilitate this interaction. It has been suggested that the periphery signals the DLN by “remote control” (Baekkevold et al., 2001; Palframan et al., 2001; Huang et al., 2008). For example, a chemokine, monocyte chemoattractant protein-1, has been suggested to drain via afferent lymphatics into the DLN (Palframan et al., 2001). A potential peripheral source of immunomodulatory mediators is the mast cell (MC), a cell type which is strategically situated beneath epithelial barriers. In addition to their presence at potential sites of pathogen entry, MCs express numerous pattern recognition and other receptors for rapidly recognizing pathogens (Prodeus et al., 1997; Malaviya et al., 1999; Supajatura et al., 2001), as well as the capacity to release large amounts of inflammatory mediators, many of which are concentrated within prominent cytoplasmic granules and are available for immediate release. One such prestored mediator is TNF (Gordon and Galli, 1990), a multifunctional cytokine which is implicated in modulating immune cell trafficking. Recent studies have shown that as rapidly as 1 h after peripheral MC activation, increased levels of TNF are detected in prenodal lymph (Frangogiannis et al., 1998) and in the DLNs (McLachlan et al., 2003). The lack of any corresponding change in DLN TNF message suggested that it was not newly synthesized locally but was derived from peripheral sources (McLachlan et al., 2003). Because a significant period of time is required for the secretion of de novo cytokine after stimulation, this peripheral source appears to be preformed MC TNF, a conclusion supported by the finding that most MCs in the vicinity of infection were degranulated (McLachlan et al., 2003). One might presume that TNF, secreted by peripheral MCs into the extracellular fluid, enters lymphatic vessels and then is carried to DLNs as a soluble molecule in the lymph. However, considering the route of trafficking and the significant distances involved, it is unclear how peripherally derived TNF could reach the DLNs without degradation, dilution to ineffective concentrations, or interaction with extracellular matrix components. This scenario is especially difficult to rationalize early in the response when it is expected that only small amounts of TNF are elaborated, and because TNF has a very short half-life in vivo (Rampart et al., 1989). This uncertainty is supported by the observation that although peripherally injected chemokines can be detected in DLNs, it typically requires the application of large quantities of recombinant protein (Stein et al., 2000; Baekkevold et al., 2001).One conceivable explanation for the long-distance action of MC-derived TNF is its trafficking in a form resistant to dilution and degradation. To investigate this possibility, we examined the process of MC exocytosis in detail. When MCs undergo secretory exocytosis of granules (degranulation), in addition to releasing some prestored mediators as freely soluble molecules (e.g., histamine and β-hexosaminidase), they also release distinct insoluble granular particles, comprised primarily of heparin proteoglycans and positively charged proteases (Schwartz et al., 1981). Other than isolated studies describing their uptake by phagocytic cells (Welsh and Geer, 1959; Miyata and Takaya, 1985), the fate of these particles after MC degranulation is largely unknown. MC granule particles are insoluble because of the large numbers of electrostatic interactions between the highly cationic MC-specific proteases and highly anionic heparin proteoglycans (Schwartz et al., 1981). Because heparin is also known to bind strongly to a variety of extracellular signaling proteins (Roberts et al., 1988; Webb et al., 1993), it is probable that some MC-derived mediators are borne within the particle structure even after their release. This possibility led us to hypothesize that MC particles might function as extracellular chaperones for MC mediators (e.g., TNF) in vivo, carrying these signaling molecules from the periphery to the DLN.     

Autologous mesenchymal stem cell–derived dopaminergic neurons function in parkinsonian macaques

A cell-based therapy for the replacement of dopaminergic neurons has been a long-term goal in Parkinson’s disease research. Here, we show that autologous engraftment of A9 dopaminergic neuron-like cells induced from mesenchymal stem cells (MSCs) leads to long-term survival of the cells and restoration of motor function in hemiparkinsonian macaques. Differentiated MSCs expressed markers of A9 dopaminergic neurons and released dopamine after depolarization in vitro. The differentiated autologous cells were engrafted in the affected portion of the striatum. Animals that received transplants showed modest and gradual improvements in motor behaviors. Positron emission tomography (PET) using [¹¹C]-CFT, a ligand for the dopamine transporter (DAT), revealed a dramatic increase in DAT expression, with a subsequent exponential decline over a period of 7 months. Kinetic analysis of the PET findings revealed that DAT expression remained above baseline levels for over 7 months. Immunohistochemical evaluations at 9 months consistently demonstrated the existence of cells positive for DAT and other A9 dopaminergic neuron markers in the engrafted striatum. These data suggest that transplantation of differentiated autologous MSCs may represent a safe and effective cell therapy for Parkinson’s disease.     

T cell–independent B cell activation induces immunosuppressive sialylated IgG antibodies

Antigen-specific Abs are able to enhance or suppress immune responses depending on the receptors that they bind on immune cells. Recent studies have shown that pro- or antiinflammatory effector functions of IgG Abs are also regulated through their Fc N-linked glycosylation patterns. IgG Abs that are agalactosylated (non-galactosylated) and asialylated are proinflammatory and induced by the combination of T cell–dependent (TD) protein antigens and proinflammatory costimulation. Sialylated IgG Abs, which are immunosuppressive, and Tregs are produced in the presence of TD antigens under tolerance conditions. T cell–independent (TI) B cell activation via B cell receptor (BCR) crosslinking through polysaccharides or via BCR and TLR costimulation also induces IgG Abs, but the Fc glycosylation state of these Abs is unknown. We found in mouse experiments that TI immune responses induced suppressive sialylated IgGs, in contrast to TD proinflammatory Th1 and Th17 immune responses, which induced agalactosylated and asialylated IgGs. Transfer of low amounts of antigen-specific sialylated IgG Abs was sufficient to inhibit B cell activation and pathogenic immune reactions. These findings suggest an immune regulatory function for TI immune responses through the generation of immunosuppressive sialylated IgGs and may provide insight on the role of TI immune responses during infection, vaccination, and autoimmunity.