Methotrexate-Related Response on Human Peripheral Blood Mononuclear Cells May Be Modulated by the Ala16Val-SOD2 Gene Polymorphism

Methotrexate (MTX) is a folic acid antagonist used in high doses as an anti-cancer treatment and in low doses for the treatment of some autoimmune diseases. MTX use has been linked to oxidative imbalance, which may cause multi-organ toxicities that can be attenuated by antioxidant supplementation. Despite the oxidative effect of MTX, the influence of antioxidant gene polymorphisms on MTX toxicity is not well studied. Therefore, we analyzed here whether a genetic imbalance of the manganese-dependent superoxide dismutase (SOD2) gene could have some impact on the MTX cytotoxic response. An in vitro study using human peripheral blood mononuclear cells (PBMCs) obtained from carriers with different Ala16Val-SOD2 genotypes (AA, VV and AV) was carried out, and the effect on cell viability and proliferation was analyzed, as well as the effect on oxidative, inflammatory and apoptotic markers. AA-PBMCs that present higher SOD2 efficiencies were more resistance to high MTX doses (10 and 100 µM) than were the VV and AV genotypes. Both lipoperoxidation and ROS levels increased significantly in PBMCs exposed to MTX independent of Ala16Val-SOD2 genotypes, whereas increased protein carbonylation was observed only in PBMCs from V allele carriers. The AA-PBMCs exposed to MTX showed decreasing SOD2 activity, but a concomitant up regulation of the SOD2 gene was observed. A significant increase in glutathione peroxidase (GPX) levels was observed in all PBMCs exposed to MTX. However, this effect was more intense in AA-PBMCs. Caspase-8 and -3 levels were increased in cells exposed to MTX, but the modulation of these genes, as well as that of the Bax and Bcl-2 genes involved in the apoptosis pathway, presented a modulation that was dependent on the SOD2 genotype. MTX at a concentration of 10 µM also increased inflammatory cytokines (IL-1β, IL-6, TNFα and Igγ) and decreased the level of IL-10 anti-inflammatory cytokine, independent of SOD2 genetic background. The results suggest that potential pharmacogenetic effect on the cytotoxic response to MTX due differential redox status of cells carriers different SOD2 genotypes.     

High-dose leptin activates human leukocytes via receptor expression on monocytes

Leptin is capable of modulating the immune response. Proinflammatory cytokines induce leptin production, and we now demonstrate that leptin can directly activate the inflammatory response. RNA expression for the leptin receptor (Ob-R) was detectable in human PBMCs. Ob-R expression was examined at the protein level by whole blood flow cytometry using an anti-human Ob-R mAb 9F8. The percentage of cells expressing leptin receptor was 25 +/- 5% for monocytes, 12 +/- 4% for neutrophils, and 5 +/- 1% for lymphocytes (only B lymphocytes). Incubation of resting PBMCs with leptin induced rapid expression of TNF-alpha and IL-6 mRNA and a dose-dependent production of TNF-alpha and IL-6 by monocytes. Incubation of resting PBMCs with high-dose leptin (250 ng/ml, 3-5 days) induced proliferation of resting cultured PBMCs and their secretion of TNF-alpha (5-fold), IL-6 (19-fold), and IFN-gamma (2.5-fold), but had no effect on IL-4 secretion. The effect of leptin was distinct from, and additive to, that seen after exposure to endotoxin or activation by the mixed lymphocyte reaction. In conclusion, Ob-R is expressed on human circulating leukocytes, predominantly on monocytes. At high doses, leptin induces proinflammatory cytokine production by resting human PBMCs and augments the release of these cytokines from activated PBMCs in a pattern compatible with the induction of Th1 cytokines. These results demonstrate that leptin has a direct effect on the generation of an inflammatory response. This is of relevance when considering leptin therapy and may partly explain the relationship among leptin, proinflammatory cytokines, insulin resistance, and obesity.     

IL-4 differentially regulates eotaxin and MCP-4 in lung epithelium and circulating mononuclear cells

To investigate the mechanisms of eosinophil recruitment in allergic airway inflammation, we examined the effects of interleukin (IL)-4, a Th2-type cytokine, on eotaxin and monocyte chemoattractant protein-4 (MCP-4) expression in human peripheral blood mononuclear cells (PBMCs; n = 10), in human lower airway mononuclear cells (n = 5), in the human lung epithelial cell lines A549 and BEAS-2B, and in human cultured airway epithelial cells. IL-4 inhibited eotaxin and MCP-4 mRNA expression induced by IL-1 beta and tumor necrosis factor-alpha in PBMCs but did not significantly inhibit expression in epithelial cells. Eotaxin and MCP-4 mRNA expression was not significantly induced by proinflammatory cytokines in lower airway mononuclear cells. IL-1 beta-induced eotaxin and MCP-4 protein production was also inhibited by IL-4 in PBMCs, whereas IL-4 enhanced eotaxin protein production in A549 cells. In contrast, dexamethasone inhibited eotaxin and MCP-4 expression in both PBMCs and epithelial cells. The divergent effects of IL-4 on eotaxin and MCP-4 expression between PBMCs and epithelial cells may create chemokine concentration gradients between the subepithelial layer and the capillary spaces that may promote the recruitment of eosinophils to the airway in Th2-type responses.     

Evidence for a superoxide permeability pathway in endosomal membranes

The compartmentalized production of superoxide (*O(2)(-)) by endosomal NADPH oxidase is important in the redox-dependent activation of NF-kappaB following interleukin 1beta (IL-1beta) stimulation. It remains unclear how *O(2)(-) produced within endosomes facilitates redox-dependent signaling events in the cytoplasm. We evaluated *O(2)(-) movement out of IL-1beta-stimulated endosomes and whether SOD1 at the endosomal surface mediates redox-signaling events required for NF-kappaB activation. The relative outward permeability of NADPH-dependent *O(2)(-) from fractionated endosomes was assessed using membrane-permeable (luminol and lucigenin) and -impermeable (isoluminol) luminescent probes for *O(2)(-). In these studies, approximately 60% of *O(2)(-) efflux out of endosomes was inhibited by treatment with either of two anion channel blockers, 4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) or niflumic acid (NFA). Furthermore, radioisotopic electrodiffusion flux assays on endomembrane proteoliposomes suggested that *O(2)(-) and Cl(-) are transported through the same DIDS-sensitive channel(s). Rab5-based immunoaffinity isolation of IL-1beta-stimulated early endosomes demonstrated SOD1 recruitment to endosomes harboring the IL-1 receptor. Finally, SOD1-deficient cells were found to be defective in their ability to activate NF-kappaB following IL-1beta stimulation. Together, these results suggest that *O(2)(-) exits endosomes through a DIDS-sensitive chloride channel(s) and that SOD1-mediated dismutation of *O(2)(-) at the endosomal surface may produce the localized H(2)O(2) required for redox-activation of NF-kappaB.     

Manganese superoxide dismutase dimorphism relationship with severity and prognosis in cardiogenic shock due to dilated cardiomyopathy

The aim was to determine (a) Ala-16Val-SOD2 dimorphisms; (b) allelic frequency and phenotype of a common Pro-Leu polymorphism in GPx1, in a cohort of patients with a cardiogenic shock (CS) due to dilated cardiomyopathy without acute coronary syndrome. Consecutive patients with de novo CS that worsened a dilated (DCM) or ischemic (ICM) cardiomyopathy. Congenital heart disease, pacemaker and other shock aetiologies were excluded. To determine oxidative stress (OS), this study evaluated lipid peroxidation, protein oxidation and erythrocyte GPx, SOD and catalase activities. Ala16Val-SOD2 (dbSNP: rs4880) and Pro198Leu-GPx1 (dbSNP: rs1050450) polymorphisms were studied by allelic discrimination using fluorogenic probes and the 5'nuclease (TaqMan) assay. Twenty-four patients (with ICM (n = 8) or DCM (n = 16), age = 57.5 ± 10.7 years, LVEF = 25.3 ± 8.5%, NT-proBNP levels = 8540 ± 1703 ng/L) were included during a 15 month follow-up. OS parameters were significantly higher in patients than in controls. Distribution of MnSOD genotypes was 47% Val/Val-variant, 29.5% Ala/Val and 23.5% Ala/Ala-variants. Severity of CS was more important in patients with Val/Val-variant and can be put in parallel with NT-proBNP levels (Val/Val-variant: 11 310 ± 3875 ng/L vs Ala/Ala-variant: 6486 ± 1375 ng/L and Ala/Val-variant: 6004 ± 2228 ng/L; p < 0.05) and hemodynamic support duration (144.6 vs Ala/Val-variant: 108.8 h and Ala/Ala-variant: 52.5 h; p < 0.05) with a positive correlation (Spearman rho = 0.72, p < 0.05). Moreover, Val/Val-variant significantly influenced the mortality (Spearman rho = 0.67, p < 0.05), but not the morbidity (p = 0.3). Distribution of GPx genotypes was 64% Pro/Pro, 18% Pro/Leu and 18% Leu/Leu. GPx-variants influenced neither GPx activities nor cardiac events. In conclusion, CS was associated with markers of increased OS. GPx polymorphism did not influence the GPx activity. Only the Val-encoding MnSOD allele was significantly correlated with the severity and prognosis of CS.     

Vitamin D(3) down-regulates proinflammatory cytokine response to Mycobacterium tuberculosis through pattern recognition receptors while inducing protective cathelicidin production

A well-known association between vitamin D(3) and infection with Mycobacterium tuberculosis has previously been reported, but little is known regarding the underlying mechanisms. We have investigated how 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] affects the proinflammatory cytokine production induced by M. tuberculosis. Furthermore, we explored whether 1,25(OH)(2)D(3) influence the production of the protective antimycobacterial peptide cathelicidin. Upon in vitro stimulation with M. tuberculosis, 1,25(OH)(2)D(3) induced a dose-dependent down-regulation of IL-6, TNFα and IFNγ, while increasing the production of IL-10 in culture supernatant as well as cathelicidin mRNA expression. This effect on cytokine response was not due to modulation of T-helper cell differentiation, as T-bet, GATA3, Foxp3 and ROR-γt mRNA expression remained unaffected. Similarly, 1,25(OH)(2)D(3) did not affect suppressor of cytokine signaling (SOCS)1 and SOCS3 mRNA expression. The mechanism whereby 1,25(OH)(2)D(3) inhibited the proinflammatory cytokine response was through reduced expression of the pattern recognition receptors (PRR) - TLR2, TLR4, Dectin-1 and mannose receptor, whose mRNA and protein expression were both reduced. The suppression of PRRs could be restored by a VDR antagonist. Upon M. tuberculosis stimulation, 1,25(OH)(2)D(3) modulates the balance in cytokine production towards an anti-inflammatory profile by repression of TLR2, TLR4, Dectin-1 and mannose receptor expression, while increasing cathelicidin production. These two effects may have beneficial consequences, by reducing the collateral tissue damage induced by proinflammatory cytokines, while the antibacterial effects of cathelicidin are enhanced.     

Bruton's tyrosine kinase is required for TLR2 and TLR4-induced TNF, but not IL-6, production

Bruton's tyrosine kinase (Btk), the gene mutated in the human immunodeficiency X-linked agammaglobulinemia, is activated by LPS and is required for LPS-induced TNF production. In this study, we have investigated the role of Btk both in signaling via another TLR (TLR2) and in the production of other proinflammatory cytokines such as IL-1beta, IL-6, and IL-8. Our data show that in X-linked agammaglobulinemia PBMCs, stimulation with TLR4 (LPS) or TLR2 (N-palmitoyl-S-[2, 3-bis(palmitoyloxy)-(2R)-propyl]-(R)-cysteine) ligands produces significantly less TNF and IL-1beta than in normal controls. In contrast, a lack of Btk has no impact on the production of IL-6, IL-8, or the anti-inflammatory cytokine, IL-10. Our previous data suggested that Btk lies within a p38-dependent pathway that stabilizes TNF mRNA. Accordingly, TaqMan quantitative PCR analysis of actinomycin D time courses presented in this work shows that overexpression of Btk is able to stabilize TNF, but not IL-6 mRNA. Furthermore, using the p38 inhibitor SB203580, we show that the TLR4-induced production of TNF, but not IL-6, requires the activity of p38 MAPK. These data provide evidence for a common requirement for Btk in TLR2- and TLR4-mediated induction of two important proinflammatory cytokines, TNF and IL-1beta, and reveal important differences in the TLR-mediated signals required for the production of IL-6, IL-8, and IL-10.     

Effects of 47C allele (rs4880) of the SOD2 gene in the production of intracellular reactive species in peripheral blood mononuclear cells with and without lipopolysaccharides induction

Abstract

Challenging of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharides (LPS) has been shown to activate monocytes and macrophages, leading to the production of pro-inflammatory cytokines and reactive oxygen species (ROS). Manganese superoxide dismutase (MnSOD) is an important enzyme that may play a central role in the response to oxidative stress. 47C> T SNP of the SOD2 gene, the -9Val MnSOD is less efficient than the -9Ala version. We have previously characterized the cellular redox status of human PBMCs expressing either -9Ala (CC) or -9Val (TT) SOD2 and analyzed the responses of these cells to oxidative stress induced by LPS. Due to the observed alterations in the activities of these antioxidant enzymes, we decided to investigate their immunocontent and analyze the production of intracellular oxidants, as well as any resulting DNA damage. PBMCs were isolated from the blood of 30 healthy human volunteers (15 volunteers per allele). We then analyzed levels of nitrite, DNA damage by comet assay, TNF-α, carboxymethyl lysine and nitrotyrosine and assessed production of intracellular reactive species by the DCFH-DA-based assay and western blots were used to analyze protein levels. Our results show that there occurs an increase in nitric oxide production in both allele groups after challenge with LPS. A significant increase in DNA damage was observed in PBMCs after an 8-h LPS challenge. Cells expressing the SOD2 47C allele quickly adapt to a more intense metabolism by upregulating cellular detoxification mechanisms. However, when these cells are stressed over a long period, they accumulate a large quantity of toxic metabolic byproducts.     

Suppressing IL-32 in monocytes impairs the induction of the proinflammatory cytokines TNFα and IL-1β

Targeting major proinflammatory cytokines such as IL-1β and TNFα is of great interest in patients with chronic inflammatory diseases, including rheumatoid arthritis, colitis, and psoriasis. The cytokine Interleukin (IL)-32 induces proinflammatory cytokines such as TNFα, IL-1β, IL-6, and chemokines. We previously used an IL-32 ligand-affinity column to purify proteinase 3, which is abundantly expressed in neutrophil and monocytic leukocytes but not in other cell types, and found that IL-32 is mainly produced by monocytic leukocytes. This evidence suggested that silencing endogenous IL-32 by short hairpin RNA (shRNA) in monocytic cells might reveal the precise function of endogenous IL-32. Indeed, lipopolysaccharide (LPS)- or phorbol myristate acetate (PMA)-induced proinflammatory cytokine production was significantly inhibited in shRNA/IL-32 stable clones as compared to control clones. Furthermore, macrophages in PMA-differentiated shRNA/IL-32 stable clones displayed remarkably impaired LPS- and IL-1β-induced proinflammatory cytokine production. These data suggest that IL-32 is not only involved in host defense against pathogens, but also might play a role in chronic inflammatory diseases. IL-32 production leads to major proinflammatory cytokine production during the initial immune response.     

TNF-alpha and H2O2 induce IL-18 and IL-18R beta expression in cardiomyocytes via NF-kappa B activation

Myocardial ischemia/reperfusion is characterized by oxidative stress and induction of proinflammatory cytokines. Interleukin (IL)-18, a member of the IL-1 family, acts as a proinflammatory cytokine, and is induced during various immune and inflammatory disorders. Therefore, in the present study we investigated whether IL-18 expression is regulated by cytokines and oxidative stress in cardiomyocytes. TNF-alpha induced rapid and sustained activation of NF-kappaB whereas H(2)O(2) induced delayed and transient activation. Both TNF-alpha and H(2)O(2) induced IL-18 mRNA and precursor protein in cardiomyocytes, and IL-18 release into culture supernatants. However, only TNF-alpha led to sustained expression. Expression of IL-18Rbeta, but not alpha, was induced by both agonists. TNF-alpha and H(2)O(2) induced delayed expression of IL-18 BP. Pretreatment with PDTC attenuated TNF-alpha and H(2)O(2) induced IL-18 and IL-18Rbeta, but not basal expression of IL-18Ralpha. These results indicate that adult cardiomyocytes express IL-18 and its receptors, and proinflammatory cytokines and oxidative stress regulate their expression via activation of NF-kappaB. Presence of both ligand and receptors suggests IL-18 impacts myocardial biology through an autocrine pathway.     

Mitochondrial respiratory chain and thioredoxin reductase regulate intermembrane Cu,Zn-superoxide dismutase activity: implications for mitochondrial energy metabolism and apoptosis

IMS (intermembrane space) SOD1 (Cu/Zn-superoxide dismutase) is inactive in isolated intact rat liver mitochondria and is activated following oxidative modification of its critical thiol groups. The present study aimed to identify biochemical pathways implicated in the regulation of IMS SOD1 activity and to assess the impact of its functional state on key mitochondrial events. Exogenous H2O2 (5 microM) activated SOD1 in intact mitochondria. However, neither H2O2 alone nor H2O2 in the presence of mitochondrial peroxiredoxin III activated SOD1, which was purified from mitochondria and subsequently reduced by dithiothreitol to an inactive state. The reduced enzyme was activated following incubation with the superoxide generating system, xanthine and xanthine oxidase. In intact mitochondria, the extent and duration of SOD1 activation was inversely correlated with mitochondrial superoxide production. The presence of TxrR-1 (thioredoxin reductase-1) was demonstrated in the mitochondrial IMS by Western blotting. Inhibitors of TxrR-1, CDNB (1-chloro-2,4-dinitrobenzene) or auranofin, prolonged the duration of H2O2-induced SOD1 activity in intact mitochondria. TxrR-1 inactivated SOD1 purified from mitochondria in an active oxidized state. Activation of IMS SOD1 by exogenous H2O2 delayed CaCl2-induced loss of transmembrane potential, decreased cytochrome c release and markedly prevented superoxide-induced loss of aconitase activity in intact mitochondria respiring at state-3. These findings suggest that H2O2, superoxide and TxrR-1 regulate IMS SOD1 activity reversibly, and that the active enzyme is implicated in protecting vital mitochondrial functions.     

The effect of fish oil supplementation on cytokine production in children

The ex vivo production of inflammatory cytokines during fish oil supplementation (n-3 polyunsaturated fatty acids, n-3 PUFA) is a matter of considerable controversy. Studies on human subjects have generally reported decreased lymphocyte proliferation and decreased production of IL-2, interferon-gamma, IL-1beta, IL-6 and TNF-alpha, but other studies showed no effect or even increased production. There are no published reports on ex vivo cytokine production in children on long-term, n-3 PUFA supplementation. The current double-blind study explored cytokine production by peripheral blood mononuclear cells (PBMCs), with and without lipopolysaccharide (LPS) stimulation in children on 12 weeks' supplementation with 300 mg/day of n-3 PUFA. Twenty-one children (aged 8-12 years) were randomized to receive 1 g canola oil (control) or 300 mg n-3 PUFA + 700 mg canola oil in a chocolate spread. Blood was then drawn and PBMCs were separated and cultured for 24 h in a culture medium with or without 10 microg/mL LPS for 5 x 10(6) PBMCs. The pro-inflammatory cytokines, IL-1beta, TNF-alpha and IL-6, and the anti-inflammatory cytokines, IL-10 and IL-1RA, were evaluated by ELISA. The levels of all the cytokines were higher in non-stimulated and LPS-stimulated cultures, from n-3 PUFA-treated subjects as compared to controls. There was no difference in the IL-1beta/IL-1RA ratio between the two groups, with and without LPS stimulation. Nevertheless, the ratio tended to be lower in the treated subjects on both occasions. In conclusion, our results indicate an increased production of both pro-inflammatory and anti-inflammatory cytokines, with and without LPS stimulation, in children on 12 weeks' n-3 PUFA supplementation.     

Mitochondrial manganese-superoxide dismutase expression in ovarian cancer: role in cell proliferation and response to oxidative stress

Superoxide dismutases (SODs) are important antioxidant enzymes responsible for the elimination of superoxide radical (O(2)(-)). The manganese-containing SOD (Mn-SOD) has been suggested to have tumor suppressor function and is located in the mitochondria where the majority of O(2)(-) is generated during respiration. Although increased reactive oxygen species (ROS) in cancer cells has long been recognized, the expression of Mn-SOD in cancer and its role in cancer development remain elusive. The present study used a human tissue microarray to analyze Mn-SOD expression in primary ovarian cancer tissues, benign ovarian lesions, and normal ovary epithelium. Significantly higher levels of Mn-SOD protein expression were detected in the malignant tissues compared with normal tissues (p < 0.05). In experimental systems, suppression of Mn-SOD expression by small interfering RNA caused a 70% increase of superoxide in ovarian cancer cells, leading to stimulation of cell proliferation in vitro and more aggressive tumor growth in vivo. Furthermore, stimulation of mitochondrial O(2)(-) production induced an increase of Mn-SOD expression. Our findings suggest that the increase in Mn-SOD expression in ovarian cancer is a cellular response to intrinsic ROS stress and that scavenging of superoxide by SOD may alleviate the ROS stress and thus reduce the simulating effect of ROS on cell growth.