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Extracellular superoxide dismutase (SOD3), an enzyme mediating dismutation of superoxide into hydrogen peroxide, has been shown to reduce inflammation by inhibiting macrophage migration into injured tissues. In inflamed tissues, superoxide is produced by the phagocytic NOX2 complex, which consists of the catalytic subunit NOX2 and several regulatory subunits (e.g., NCF1). To analyze whether SOD3 can regulate inflammation in the absence of functional NOX2 complex, we injected an adenoviral vector overexpressing SOD3 directly into the arthritic paws of Ncf1∗/∗ mice with collagen-induced arthritis. SOD3 reduced arthritis severity in both oxidative burst-deficient Ncf1∗/∗ mice and also in wild-type mice. The NOX2 complex independent anti-inflammatory effect of SOD3 was further characterized in peritonitis, and SOD3 was found to reduce macrophage infiltration independently of NOX2 complex functionality. We conclude that the SOD3-mediated anti-inflammatory effect on arthritis and peritonitis operates independently of NOX2 complex derived oxidative burst.

Summary

The extracellular isoform of superoxide dismutase (EC-SOD, Sod3) plays a protective role against various diseases and injuries mediated by oxidative stress. To investigate the pathophysiological roles of EC-SOD, we generated tetracycline-inducible Sod3 transgenic mice and directed the tissue-specific expression of transgenes by crossing Sod3 transgenic mice with tissue-specific transactivator transgenics. Double transgenic mice with liver-specific expression of Sod3 showed increased EC-SOD levels predominantly in the plasma as the circulating form, whereas double transgenic mice with neuronal-specific expression expressed higher levels of EC-SOD in hippocampus and cortex with intact EC-SOD as the dominant form. EC-SOD protein levels also correlated well with increased SOD activities in double transgenic mice. In addition to enabling tissue-specific expression, the transgene expression can be quickly turned on and off by doxycycline supplementation in the mouse chow. This mouse model, thus, provides the flexibility for on–off control of transgene expression in multiple target tissues.

Increased reactive oxygen species (ROS) such as superoxide have been implicated as causal elements of oncogenesis. A variety of cancers have displayed changes in steady state levels of key antioxidant enzymes with the mitochondrial form of superoxide dismutase (MnSOD) being commonly implicated. Increasing MnSOD expression suppresses the malignant phenotype in various cancer cell lines and suppresses tumor formation in xenograft and transgenic mouse models. We examined the impact of MnSOD expression in the development of T cell lymphoma in mice expressing pro-apoptotic Bax. Lck-Bax38/1 transgenic mice were crossed to mice overexpressing MnSOD (Lck-MnSOD) as well as MnSOD +/− mice. The effect of MnSOD on apoptosis, cell cycle, chromosomal instability (CIN), and lymphoma development was determined. The apoptotic and cell cycle phenotypes observed in thymocytes from control and Bax transgenic mice were unaffected by variations in MnSOD levels. Remarkably, increased gene dosage of MnSOD significantly decreased aneuploidy in pre-malignant thymocytes as well as the onset of tumor formation in Lck-Bax38/1 mice. The observed effects of MnSOD support a role for ROS in CIN and tumor formation in this mouse model of T cell lymphoma.

Both rheumatoid arthritis and animal models of autoimmune arthritis are characterized by hyperactivation of synovial cells and hyperplasia of the synovial membrane. The activated synovial cells produce inflammatory cytokines and degradative enzymes that lead to destruction of cartilage and bones. Effective treatment of arthritis may require elimination of most or all activated synovial cells. The death factor Fas/Apo-1 and its ligand (FasL) play pivotal roles in maintaining self-tolerance and immune privilege. Fas is expressed constitutively in most tissues, and is dramatically upregulated at the site of inflammation. In both rheumatoid arthritis and animal models of autoimmune arthritis, high levels of Fas are expressed on activated synovial cells and infiltrating leukocytes in the inflamed joints. Unlike Fas, however, the levels of FasL expressed in the arthritic joints are extremely low, and most activated synovial cells survive despite high levels of Fas expression. To upregulate FasL expression in the arthritic joints, we have generated a recombinant replication-defective adenovirus carrying FasL gene; injection of the FasL virus into inflamed joints conferred high levels of FasL expression, induced apoptosis of synovial cells, and ameliorated collagen-induced arthritis in DBA/1 mice. The Fas-ligand virus also inhibited production of interferon-gamma by collagen-specific T cells. Coadministration of Fas-immunoglobulin fusion protein with the Fas-ligand virus prevented these effects, demonstrating the specificity of the Fas-ligand virus. Thus, FasL gene transfer at the site of inflammation effectively ameliorates autoimmune disease.

Mutations in superoxide dismutase 1 (SOD1, EC 1.15.1.1) cause familial amyotrophic lateral sclerosis (fALS); with aggregated forms of mutant protein accumulating in spinal cord tissues of transgenic mouse models and human patients. Mice over-expressing wild-type human SOD1 (WT hSOD1) do not develop ALS-like disease, but co-expression of WT enzyme at high levels with mutant SOD1 accelerates the onset of motor neuron disease compared to mice expressing mutant hSOD1 alone. Spinal cords of mice expressing both proteins contain aggregated forms of mutant protein and, in some cases, evidence of co-aggregation of WT hSOD1 enzyme. In the present study, we used a cell culture model of mutant SOD1 aggregation to examine how the presence of WT SOD1 affects mutant protein aggregation, finding that co-expression of WT SOD1, human (hSOD1) or mouse (mSOD1), delayed the formation of mutant hSOD1 aggregates; in essence appearing to slow the aggregation rate. In some combinations of WT and mutant hSOD1 co-expression, the aggregates that did eventually form appeared to contain WT hSOD1 protein. However, WT mSOD1 did not co-aggregate with mutant hSOD1 despite displaying a similar ability to slow mutant hSOD1 aggregation. Together, these studies indicate that WT SOD1 (human or mouse), when expressed at levels equivalent to the mutant protein, modulates aggregation of FALS-mutant hSOD1.

Inflammatory cell migration characteristic of ischemic damages has a dual role providing the tissue with factors needed for tissue injury recovery simultaneously causing deleterious development depending on the quality and the quantity of infiltrated cells. Extracellular superoxide dismutase (SOD3) has been shown to have an anti-inflammatory role in ischemic injuries where it increases the recovery process by activating mitogen signal transduction and increasing cell proliferation. However, SOD3 derived effects on inflammatory cytokine and adhesion molecule expression, which would explain reduced inflammation in vascular lesions, has not been properly characterized. In the present work the effect of SOD3 on the inflammatory cell extravasation was studied in vivo in rat hind limb ischemia and mouse peritonitis models by identifying the migrated cells and analyzing SOD3-derived response on inflammatory cytokine and adhesion molecule expression. SOD3 overexpression significantly reduced TNFα, IL1α, IL6, MIP2, and MCP-1 cytokine and VCAM, ICAM, P-selectin, and E-selectin adhesion molecule expressions in injured tissues. Consequently the mononuclear cell, especially CD68+ monocyte and CD3+ T cell infiltration were significantly decreased whereas granulocyte migration was less affected. According to our data SOD3 has a selective anti-inflammatory role in ischemic damages preventing the migration of reactive oxygen producing monocyte/macrophages, which in excessive amounts could potentially further intensify the tissue injuries therefore suggesting potential for SOD3 in treatment of inflammatory disorders.

Backgrounds and Purpose

Superoxide is associated with spontaneous intracerebral hemorrhage (ICH) during hypertension. The goal of this study was to test the hypothesis that changes in superoxide, in genetically altered mice with deletion and overexpression of CuZn-superoxide dismutase (SOD1), modulate susceptibility to ICH.

Methods

Chronic hypertension was produced by infusion of angiotensin II (AngII) and an inhibitor of nitric oxide synthase in drinking water in SOD1 transgenic (SOD1Tg) mice, SOD1 deficient (SOD1−/−) mice, and their respective wild-type (WT)-littermates. Acute hypertension was produced by daily injections of AngII in some mice with chronic hypertension to produce ICH. We evaluated susceptibility to ICH, oxidative stress [superoxide, NAD(P)H oxidase activity, SOD activity], gene expression, and activity of matrix metalloproteinases (MMPs).

Results

Incidence, size, and number of ICH were reduced in SOD1Tg mice, and were increased in SOD1−/− mice compared to their WT-littermates. Levels of superoxide increased in the brain even before developing ICH in WT-littermates, while levels of superoxide remained low in SOD1Tg mice. Changes in level of MMP-9 paralleled oxidative stress in SOD1Tg mice and WT-littermates. Moreover, levels of superoxide and MMP-9 were greater in SOD1−/− mice than WT-littermates after induction of ICH. Active MMPs colocalized on cerebral vessels that appeared to lead toward regions with ICH.

Conclusions

These results suggest that superoxide contributes to the pathogenesis of spontaneous ICH, possibly through activation of MMP-9, and that SOD1 protects against spontaneous ICH during hypertension.

In familial and sporadic amyotrophic lateral sclerosis (ALS) and in rodent models of the disease, alterations in the ubiquitin-proteasome system (UPS) may be responsible for the accumulation of potentially harmful ubiquitinated proteins, leading to motor neuron death. In the spinal cord of transgenic mice expressing the familial ALS superoxide dismutase 1 (SOD1) gene mutation G93A (SOD1G93A), we found a decrease in constitutive proteasome subunits during disease progression, as assessed by real-time PCR and immunohistochemistry. In parallel, an increased immunoproteasome expression was observed, which correlated with a local inflammatory response due to glial activation. These findings support the existence of proteasome modifications in ALS vulnerable tissues. To functionally investigate the UPS in ALS motor neurons in vivo, we crossed SOD1G93A mice with transgenic mice that express a fluorescently tagged reporter substrate of the UPS. In double-transgenic UbG76V-GFP /SOD1G93A mice an increase in UbG76V-GFP reporter, indicative of UPS impairment, was detectable in a few spinal motor neurons and not in reactive astrocytes or microglia, at symptomatic stage but not before symptoms onset. The levels of reporter transcript were unaltered, suggesting that the accumulation of UbG76V-GFP was due to deficient reporter degradation. In some motor neurons the increase of UbG76V-GFP was accompanied by the accumulation of ubiquitin and phosphorylated neurofilaments, both markers of ALS pathology. These data suggest that UPS impairment occurs in motor neurons of mutant SOD1-linked ALS mice and may play a role in the disease progression.

Manganese superoxide dismutase (MnSOD) is a nuclear-encoded antioxidant enzyme that localizes to the mitochondria. Expression of MnSOD is essential for the survival of aerobic life. Transgenic mice expressing a luciferase reporter gene under the control of the human MnSOD promoter demonstrate that the level of MnSOD is reduced prior to the formation of cancer. Overexpression of MnSOD in transgenic mice reduces the incidences and multiplicity of papillomas in a DMBA/TPA skin carcinogenesis model. However, MnSOD deficiency does not lead to enhanced tumorigenicity of skin tissue similarly treated because MnSOD can modulate both the p53-mediated apoptosis and AP-1-mediated cell proliferation pathways. Apoptosis is associated with an increase in mitochondrial levels of p53 suggesting a link between MnSOD deficiency and mitochondrial-mediated apoptosis. Activation of p53 is preventable by application of a SOD mimetic (MnTE-2-PyP5+). Thus, p53 translocation to mitochondria and subsequent inactivation of MnSOD explain the observed mitochondrial dysfunction that leads to transcription-dependent mechanisms of p53-induced apoptosis. Administration of MnTE-2-PyP5+ following apoptosis but prior to proliferation leads to suppression of protein carbonyls and reduces the activity of AP-1 and the level of the proliferating cellular nuclear antigen, without reducing the activity of p53 or DNA fragmentation following TPA treatment. Remarkably, the incidence and multiplicity of skin tumors are drastically reduced in mice that receive MnTE-2-PyP5+ prior to cell proliferation. The results demonstrate the role of MnSOD beyond its essential role for survival and suggest a novel strategy for an antioxidant approach to cancer intervention.

Cytokines play key roles in spontaneous CD4+ T cell–mediated chronic autoimmune arthritis in SKG mice, a new model of rheumatoid arthritis. Genetic deficiency in IL-6 completely suppressed the development of arthritis in SKG mice, irrespective of the persistence of circulating rheumatoid factor. Either IL-1 or TNF-α deficiency retarded the onset of arthritis and substantially reduced its incidence and severity. IL-10 deficiency, on the other hand, exacerbated disease, whereas IL-4 or IFN-γ deficiency did not alter the disease course. Synovial fluid of arthritic SKG mice contained high amounts of IL-6, TNF-α, and IL-1, in accord with active transcription of these cytokine genes in the afflicted joints. Notably, immunohistochemistry revealed that distinct subsets of synovial cells produced different cytokines in the inflamed synovium: the superficial synovial lining cells mainly produced IL-1 and TNF-α, whereas scattered subsynovial cells produced IL-6. Thus, IL-6, IL-1, TNF-α, and IL-10 play distinct roles in the development of SKG arthritis; arthritogenic CD4+ T cells are not required to skew to either Th1 or Th2; and the appearance of rheumatoid factor is independent of joint inflammation. The results also indicate that targeting not only each cytokine but also each cell population secreting distinct cytokines could be an effective treatment of rheumatoid arthritis.

Pro-inflammatory cytokines and chemokines play critical roles in autoimmune diseases including rheumatoid arthritis (RA). Recently, it has been reported that β-arrestin 1 and 2 are involved in the regulation of inflammation. We hypothesized that β-arrestin 1 and 2 play critical roles in murine models of RA. Using a collagen-induced arthritis (CIA) and a human TNFα transgenic (TNFtg) mouse model, we demonstrated that β-arrestin 1 and 2 expression are significantly increased in joint tissue of CIA mice and TNFtg mice. In fibroblast-like synoviocytes (FLS) isolated from hind knee joint of CIA mice, we observed an increase of β-arrestin 1 and 2 protein and mRNA levels in the early stage of arthritis. In FLS, low molecular weight hyaluronan (HA)-induced TNFα and IL-6 production was increased by overexpression of β-arrestin 1 but decreased by overexpression of β-arrestin 2 demonstrating isoform specific regulation. TNFα and HA induced an increase of β-arrestin 1 and 2 expression in FLS, while high mobility group box (HMGB)-1 only stimulated β-arrestin 1 expression. TNFα- or HA- induced β-arrestin 2 expression was blocked by a p38 inhibitor. To examine the in vivo role of β-arrestin 2 in the pathogenesis of arthritis, WT and β-arrestin 2 KO mice were subjected to collagen antibody-induced arthritis (CAIA). β-arrestin 2 KO mice exhibited more severe arthritis in CAIA. Thus β-arrestin 2 is anti-inflammatory in CAIA. These composite observations suggest that β-arrestin 1 and 2 differentially regulate FLS inflammation and increased β-arrestin 2 may reduce experimental arthritis severity.

Background

The synovial tissue is a primary target of many inflammatory arthropathies, including psoriatic arthritis (PsA). Identification of proinflammatory molecules in the synovium may help to identify potentially therapeutic targets.

Objective

To investigate extensively the features of cell infiltration and expression of mediators of inflammation and joint destruction in the synovium of patients with PsA compared with patients with rheumatoid arthritis matched for disease duration and use of drugs.

Methods

Multiple synovial tissue biopsy specimens were obtained by arthroscopy from an inflamed joint in 19 patients with PsA (eight oligoarthritis, 11 polyarthritis) and 24 patients with rheumatoid arthritis. Biopsy specimens were analysed by immunohistochemistry to detect T cells, plasma cells, fibroblast‐like synoviocytes, macrophages, proinflammatory cytokines, matrix metalloproteinases and tissue inhibitor metalloproteinase‐1, adhesion molecules and vascular markers. Stained sections were evaluated by digital image analysis.

Results

The synovial infiltrate of patients with PsA and rheumatoid arthritis was comparable with regard to numbers of fibroblast‐like synoviocytes and macrophages. T cell numbers were considerably lower in the synovium of patients with PsA. The number of plasma cells also tended to be lower in PsA. The expression of tumour necrosis factor alpha (TNFα), interleukin (IL) 1β, IL6 and IL18 was as high in PsA as in rheumatoid arthritis. The expression of matrix metalloproteinases, adhesion molecules and vascular markers was comparable for PsA and rheumatoid arthritis.

Conclusion

These data show increased proinflammatory cytokine expression in PsA synovium, comparable to results obtained for rheumatoid arthritis, and support the notion that, in addition to TNFα blockade, there may be a rationale for treatments directed at IL1β, IL6 and IL18.

Elevated free radical generation in inflamed joints and impaired antioxidant system has been implicated in rheumatoid arthritis (RA). Green tea extracts (GTE) have been shown to reduce inflammation in inflammatory arthritis murine model. This study investigates possible mechanisms by which vitamin C and GTE protect joints in RA rat model. This study included forty adult male rats that were divided into four groups (10 rats each); control group, collagen II induced RA group (CII), CII treated with vitamin C (CII + Vit C) and CII treated with GTE (CII + GTE) in physiology laboratory, Assiut University, Egypt. After 45 days of treatment, plasma levels of lipid peroxides (LPO), nitric oxide (NO), ceruloplasmin (CP), superoxide dismutase (SOD), uric acid (UA) and glutathione (GSH) were detected using colorimetric methods, PGE2 using ELISA and copper (Cu) and zinc (Zn) using spectrometer. In CII group, levels of LPO, NO, PGE2, UA, CP, Cu were higher while SOD, GSH, Zn were lower than controls. In groups treated with vitamin C and GTE, levels of SOD, GSH were increased while levels of LPO, NO, PGE2, Cu, CP were decreased compared with CII group. Levels of UA were decreased and Zn increased in GTE treated group compared with CII group. GTE treated group showed higher Zn and low Cu levels compared with vitamin C treated group. This study suggests proper GTE and vitamin C intake may effectively normalize the impaired oxidant/antioxidant system and delaying complication of RA.

Extracellular superoxide dismutase (EC-SOD, or SOD3) is the major extracellular antioxidant enzyme in the lung. To study the biologic role of EC-SOD in hyperoxic-induced pulmonary disease, we created transgenic (Tg) mice that specifically target overexpression of human EC-SOD (hEC-SOD) to alveolar type II and nonciliated bronchial epithelial cells. Mice heterozygous for the hEC-SOD transgene showed threefold higher EC-SOD levels in the lung compared with wild-type (Wt) littermate controls. A significant amount of hEC-SOD was present in the epithelial lining fluid layer. Both Tg and Wt mice were exposed to normobaric hyperoxia (>99% oxygen) for 48, 72, and 84 hours. Mice overexpressing hEC-SOD in the airways attenuated the hyperoxic lung injury response, showed decreased morphologic evidence of lung damage, had reduced numbers of recruited inflammatory cells, and had a reduced lung wet/dry ratio. To evaluate whether reduced numbers of neutrophil infiltration were directly responsible for the tolerance to oxygen toxicity observed in the Tg mice, we made Wt and Tg mice neutropenic using anti-neutrophil antibodies and subsequently exposed them to 72 hours of hyperoxia. Both Wt and Tg neutrophil-depleted (ND) mice have less severe lung injury compared with non-ND animals, thus providing direct evidence that neutrophils recruited to the lung during hyperoxia play a distinct role in the resultant acute lung injury. We conclude that oxidative and inflammatory processes in the extracellular lung compartment contribute to hyperoxic-induced lung damage and that overexpression of hEC-SOD mediates a protective response to hyperoxia, at least in part, by attenuating the neutrophil inflammatory response.

Background

During pathology of the nervous system, increased extracellular ATP acts both as a cytotoxic factor and pro-inflammatory mediator through P2X7 receptors. In animal models of amyotrophic lateral sclerosis (ALS), astrocytes expressing superoxide dismutase 1 (SOD1G93A) mutations display a neuroinflammatory phenotype and contribute to disease progression and motor neuron death. Here we studied the role of extracellular ATP acting through P2X7 receptors as an initiator of a neurotoxic phenotype that leads to astrocyte-mediated motor neuron death in non-transgenic and SOD1G93A astrocytes.

Methods

We evaluated motor neuron survival after co-culture with SOD1G93A or non-transgenic astrocytes pretreated with agents known to modulate ATP release or P2X7 receptor. We also characterized astrocyte proliferation and extracellular ATP degradation.

Results

Repeated stimulation by ATP or the P2X7-selective agonist BzATP caused astrocytes to become neurotoxic, inducing death of motor neurons. Involvement of P2X7 receptor was further confirmed by Brilliant blue G inhibition of ATP and BzATP effects. In SOD1G93A astrocyte cultures, pharmacological inhibition of P2X7 receptor or increased extracellular ATP degradation with the enzyme apyrase was sufficient to completely abolish their toxicity towards motor neurons. SOD1G93A astrocytes also displayed increased ATP-dependent proliferation and a basal increase in extracellular ATP degradation.

Conclusions

Here we found that P2X7 receptor activation in spinal cord astrocytes initiated a neurotoxic phenotype that leads to motor neuron death. Remarkably, the neurotoxic phenotype of SOD1G93A astrocytes depended upon basal activation the P2X7 receptor. Thus, pharmacological inhibition of P2X7 receptor might reduce neuroinflammation in ALS through astrocytes.

Oxidative damage is a major cause of lung injury during systemic inflammatory response syndrome. In the present study, expression of an anti-oxidant enzyme, extracellular superoxide dismutase (EC-SOD), and its protective role against pulmonary oxidative damage were investigated using mouse models of systemic inflammation. Intraperitoneal injection with bacterial endotoxin lipopolysaccharides (LPS, 20 mg/kg) caused oxidative damage in lungs as assessed by increased tyrosine nitration in proteins. LPS administration also resulted in a rapid and significant loss of pulmonary EC-SOD more than 80% in a time- and dose-dependent manner, but other types of SODs, cytoplasmic CuZn-SOD and mitochondrial Mn-SOD, were not affected. EC-SOD protein is most abundant in lungs but also present at high levels in other tissues such as heart and white fat; however, the LPS-mediated decrease in this enzyme was most apparent in the lungs. Intravenous injection of mice with tumor necrosis factor α (10 μg per mouse) also caused 60% decrease in EC-SOD in lungs, suggesting that the EC-SOD down-regulation is mediated by this LPS-inducible inflammatory cytokine. A protective role of EC-SOD against LPS-mediated systemic inflammation was shown by an increased survival rate (75% vs.29% in 5 days) and decreased pulmonary oxidative damage in EC-SOD transgenic mice that overexpress the human EC-SOD gene. These results demonstrate that the inflammation-mediated EC-SOD down-regulation has a major pathophysiological impact during the systemic inflammatory response syndrome.

Acetylcholine, the principal vagus neurotransmitter, inhibits inflammation by suppressing the production of pro-inflammatory cytokines through a mechanism dependent on the α7 nicotinic acetylcholine receptor subunit (alpha7nAChR) that explains why vagus nerve stimulation is anti-inflammatory in nature. Strong expression of alpha7nAChR in the synovium of rheumatoid arthritis and psoriatic arthritis patients was detected. Peripheral macrophages and synovial fibroblasts respond in vitro to specific alpha7nAChR cholinergic stimulation with potent inhibition of proinflammatory cytokines. Fibroblasts balance inflammatory mechanisms and arthritis development through feedback cholinergic stimulation by nearby immune cells. Collagen induced arthritis in alpha7nAChR(-/-) mice was significantly severe and showed increased synovial inflammation and joint destruction compared to the wild-type mice. Similar to vagal nerve stimulation and alpha7nAChR agonists, polyunsaturated fatty acids: eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) also suppress inflammation. In view of their similar anti-inflammatory actions, it is proposed that vagal nerve stimulation, alpha7nAChR agonists and EPA and DHA may augment the formation of anti-inflammatory lipid molecules: lipoxins, resolvins, protectins and maresins. This implies that therapies directed at regulation of the cholinergic and alpha7nAChR mediated mechanisms and enhancing the formation of lipoxins, resolvins, protectins and maresins may halt and/or ameliorate rheumatoid arthritis, lupus and other rheumatological conditions.

Extracellular superoxide dismutase (EC-SOD) is expressed at high levels in lungs. EC-SOD has a polycationic matrix-binding domain that binds to polyanionic constituents in the matrix. Previous studies indicate that EC-SOD protects the lung in both bleomycin- and asbestos-induced models of pulmonary fibrosis. Although the mechanism of EC-SOD protection is not fully understood, these studies indicate that EC-SOD plays an important role in regulating inflammatory responses to pulmonary injury. Hyaluronan is a polyanionic high molecular mass polysaccharide found in the extracellular matrix that is sensitive to oxidant-mediated fragmentation. Recent studies found that elevated levels of low molecular mass hyaluronan are associated with inflammatory conditions. We hypothesize that EC-SOD may inhibit pulmonary inflammation in part by preventing superoxide-mediated fragmentation of hyaluronan to low molecular mass fragments. We found that EC-SOD directly binds to hyaluronan and significantly inhibits oxidant-induced degradation of this glycosaminoglycan. In vitro human polymorphic neutrophil chemotaxis studies indicate that oxidative fragmentation of hyaluronan results in polymorphic neutrophil chemotaxis and that EC-SOD can completely prevent this response. Intratracheal injection of crocidolite asbestos in mice leads to pulmonary inflammation and injury that is enhanced in EC-SOD knock-out mice. Notably, hyaluronan levels are increased in the bronchoalveolar lavage fluid after asbestos-induced pulmonary injury, and this response is markedly enhanced in EC-SOD knock-out mice. These data indicate that inhibition of oxidative hyaluronan fragmentation probably represents one mechanism by which EC-SOD inhibits inflammation in response to lung injury.

Background

Multiple cellular functions are compromised in amyotrophic lateral sclerosis (ALS). In familial ALS (FALS) with Cu/Zn superoxide dismutase (SOD1) mutations, the mechanisms by which the mutation in SOD1 leads to such a wide range of abnormalities remains elusive.

Methodology/Principal Findings

To investigate underlying cellular conditions caused by the SOD1 mutation, we explored mutant SOD1-interacting proteins in the spinal cord of symptomatic transgenic mice expressing a mutant SOD1, SOD1Leu126delTT with a FLAG sequence (DF mice). This gene product is structurally unable to form a functional homodimer. Tissues were obtained from both DF mice and disease-free mice expressing wild-type with FLAG SOD1 (WF mice). Both FLAG-tagged SOD1 and cross-linking proteins were enriched and subjected to a shotgun proteomic analysis. We identified 34 proteins (or protein subunits) in DF preparations, while in WF preparations, interactions were detected with only 4 proteins.

Conclusions/Significance

These results indicate that disease-causing mutant SOD1 likely leads to inadequate protein-protein interactions. This could be an early and crucial process in the pathogenesis of FALS.

BACKGROUND: Tissues undergoing a chronic inflammatory process, such as the synovium in rheumatoid arthritis, are characterized by the infiltration of lymphocytes of different subsets and activation of monocyte/macrophages. Interleukin-1 (IL-1), a monocyte/ macrophage product that stimulates synovial fibroblasts to produce matrix metalloproteinases (MMPs), prostaglandins, and other cytokines, also has profound effects on the synthesis of extracellular matrix components such as type I collagen. In previous studies, we have shown that synovial fibroblasts and chondrocytes isolated from human joint tissues are particularly sensitive to prostaglandins, which modulate the effects of IL-1 on collagen gene expression in an autocrine manner. MATERIALS AND METHODS: BALBc/3T3 fibroblasts were treated with IL-1 and prostaglandins in the absence and presence of indomethacin to inhibit endogenous prostaglandin biosynthesis. Collagen synthesis was analyzed by SDS-PAGE as [3H]proline-labeled, secreted proteins, and prostaglandin production and cyclic adenosine 3',5'-cyclic monophosphate (camp) content were assayed. The expression of type I collagen gene (Col1a1) promoter-reporter gene constructs was examined in transient transfection experiments, and the binding of nuclear factors to the Col1a1 promoter region spanning -222 bp/+ 116 bp was analyzed by DNase I footprinting and electrophoretic mobility shift (EMSA) assays. RESULTS: IL-1 increased the synthesis of type I and type III collagens in BALBc/3T3 fibroblasts; greater increases were observed when IL-1-stimulated synthesis of PGE2 was blocked by indomethacin. Transient transfection experiments demonstrated dose-dependent inhibition of the-222 bp Col1a1 promoter by exogenously added prostaglandins with the order of potency of PGF2alpha > PGE2 > PGE1 DNase I footprinting showed increased protection, which extended from the region immediately upstream of the TATA box, owing to the binding of nuclear factors from PGE2- or PGE1-treated BALBc/3T3 cells. EMSA analysis showed zinc-dependent differences in the binding of nuclear factors from untreated and prostaglandin-treated cells to the -84 bp/-29 bp region of the Col1a1 promoter. CONCLUSIONS: These results show that the inhibition of Col1a1 expression by IL-1 in fibroblasts is mediated by prostaglandins at the transcriptional level and suggest that PGE-responsive factors may interact directly or indirectly with basal regulatory elements in the proximal promoter region of the Col1a1 gene.

Nicotinamide phosphoribosyltransferase (NAMPT), also known as visfatin, is the rate-limiting enzyme in the salvage pathway of NAD biosynthesis from nicotinamide. Since its expression is upregulated during inflammation, NAMPT represents a novel clinical biomarker in acute lung injury, rheumatoid arthritis, and Crohn's disease. However, its role in disease progression remains unknown. We report here that NAMPT is a key player in inflammatory arthritis. Increased expression of NAMPT was confirmed in mice with collagen-induced arthritis, both in serum and in the arthritic paw. Importantly, a specific competitive inhibitor of NAMPT effectively reduced arthritis severity with comparable activity to etanercept, and decreased pro-inflammatory cytokine secretion in affected joints. Moreover, NAMPT inhibition reduced intracellular NAD concentration in inflammatory cells and circulating TNFα levels during endotoxemia in mice. In vitro pharmacological inhibition of NAMPT reduced the intracellular concentration of NAD and pro-inflammatory cytokine secretion by inflammatory cells. Thus, NAMPT links NAD metabolism to inflammatory cytokine secretion by leukocytes, and its inhibition might therefore have therapeutic efficacy in immune-mediated inflammatory disorders.

Introduction

Type 4 phosphodiesterases (PDE4) play an important role in immune cells through the hydrolysis of the second messenger, cAMP. Inhibition of PDE4 has previously been shown to suppress immune and inflammatory responses, demonstrating PDE4 to be a valid therapeutic target for immune-mediated pathologies. We assessed the anti-inflammatory effects of a novel PDE4 inhibitor, apremilast, in human synovial cells from rheumatoid arthritis (RA) patients, as well as two murine models of arthritis.

Methods

Cells liberated from tissue excised from arthritic joints of RA patients were cultured in the presence of increasing concentrations of apremilast for 48 hours and spontaneous tumour necrosis factor-alpha (TNFα) production was analysed in culture supernatants by ELISA. In addition, arthritis was induced in BALB/c and DBA/1 mice by passive transfer of anti-type II collagen mAb and immunisation with type II collagen, respectively. Mice with established arthritis received 5 or 25 mg/kg apremilast and disease severity was monitored relative to mice receiving vehicle alone. At the end of the study, paws were removed and processed for histopathological assessment. Behavioural effects of apremilast, relative to rolipram, were assessed in naïve DBA/1 mice using an automated activity monitor (LABORAS).

Results

Apremilast dose dependently inhibited spontaneous release of TNFα from human rheumatoid synovial membrane cultures. Furthermore, apremilast significantly reduced clinical score in both murine models of arthritis over a ten day treatment period and maintained a healthy joint architecture in a dose-dependent manner. Importantly, unlike rolipram, apremilast demonstrated no adverse behavioural effects in naïve mice.

Conclusions

Apremilast is an orally available PDE4 inhibitor that reduces TNFα production from human synovial cells and significantly suppresses experimental arthritis. Apremilast appears to be a potential new agent for the treatment of rheumatoid arthritis.

Mutations in human copper-zinc superoxide dismutase (SOD1) cause an inherited form of amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease, motor neuron disease). Insoluble forms of mutant SOD1 accumulate in neural tissues of human ALS patients and in spinal cords of transgenic mice expressing these polypeptides, suggesting that SOD1-linked ALS is a protein misfolding disorder. Understanding the molecular basis for how the pathogenic mutations give rise to SOD1 folding intermediates, which may themselves be toxic, is therefore of keen interest. A critical step on the SOD1 folding pathway occurs when the copper chaperone for SOD1 (CCS) modifies the nascent SOD1 polypeptide by inserting the catalytic copper cofactor and oxidizing its intrasubunit disulfide bond. Recent studies reveal that pathogenic SOD1 proteins coming from cultured cells and from the spinal cords of transgenic mice tend to be metal-deficient and/or lacking the disulfide bond, raising the possibility that the disease-causing mutations may enhance levels of SOD1-folding intermediates by preventing or hindering CCS-mediated SOD1 maturation. This mini-review explores this hypothesis by highlighting the structural and biophysical properties of the pathogenic SOD1 mutants in the context of what is currently known about CCS structure and action. Other hypotheses as to the nature of toxicity inherent in pathogenic SOD1 proteins are not covered.

Studies have demonstrated that increased oxidative stress contributes to the pathogenesis and the development of pulmonary artery hypertension (PAH). Extracellular superoxide dismutase (SOD3) is essential for removing extracellular superoxide anions and it is highly expressed in lung tissue. However, it is not clear whether endogenous SOD3 can influence the development of PAH. Here we examined the effect of SOD3 knockout on hypoxia-induced PAH in mice and a loss-of-function SOD3 gene mutation (SOD3E124D) on monocrotaline (40 mg/kg)-induced PAH in rats. SOD3 knockout significantly exacerbated 2 weeks hypoxia-induced right ventricular (RV) pressure and RV hypertrophy, while RV pressure in SOD3 KO mice under normoxic conditions is similar to wild type controls. In untreated control rats at age of 8 weeks, there was no significant difference between wild type and SOD3E124D rats in RV pressure and the ratio of RV weight to left ventricular weight (0.25±0.02 in wild type rats vs. 0.25±0.01 in SOD3E124D rats). However, monocrotaline caused significantly greater increases of RV pressure in SOD3E124D rats (48.6±1.8 mmHg in wild type vs. 57.5±3.1 mmHg in SOD3E124D rats), of the ratio of RV weight to left ventricular weight (0.41±0.01 vs. 0.50±0.09, p<0.05), and of the percentage of fully muscularized small arterioles in SOD3E124D rats (55.2±2.3 % vs. 69.9 ±2.6 %, p<0.05). Together, these findings indicate that the endogenous SOD3 has no role in the development of PAH under control conditions, but plays an important role in protecting the lung from the development of PAH under stress conditions.

Delivery of recombinant superoxide dismutase to lung is limited by its short half life and poor tissue penetration. We hypothesized that a chimeric protein, SOD2/3, containing the enzymatic domain of manganese superoxide dismutase (SOD2) and the heparan binding domain of extracellular superoxide dismutase (SOD3) would allow for the delivery of more sustained lung and pulmonary vascular antioxidant activity compared to SOD2. We administered SOD2/3 to rats by intratracheal, (IT) intraperitoneal (IP), or intravenous (IV) routes and evaluated the presence, localization and activity of lung SOD2/3 1 day later using Western blot, immunohistochemistry and SOD activity gel. The effect of IT SOD2/3 on the pulmonary and systemic circulation was studied in vivo in chronically catheterized rats exposed to acute hypoxia. Active SOD2/3 was detected in lung 1 day following IT administration but not detected following IP or IV SOD2/3 administration or IT SOD2. The physiologic response to acute hypoxia, vasoconstriction in the pulmonary circulation and vasodilation in the systemic circulation, was enhanced in rats treated 1 day earlier with IT SOD2/3. These findings indicate that IT administration of SOD2/3 effectively delivers sustained enzyme activity to the lung as well as pulmonary circulation and has a longer tissue half-life compared to native SOD2. Further testing in models of chronic lung or pulmonary vascular diseases mediated by excess superoxide should consider the longer tissue half-life of SOD2/3 as well as its potential systemic vascular effects.