Manuel Conde

Oxidative Stress Triggers STAT3 Tyrosine Phosphorylation and Nuclear Translocation in Human Lymphocytes

Oxidizing agents are powerful activators of factors responsible for the transcriptional activation of cytokine-encoding genes involved in tissue injury. In this study we show evidence that STAT3 is a transcription factor whose activity is modulated by H2O2 in human lymphocytes, in which endogenous catalase had previously been inhibited. H2O2-induced nuclear translocation of STAT3 to form sequence-specific DNA-bound complexes was evidenced by immunoblotting of nuclear fractions and electrophoretic mobility shift assays, and vanadate was found to strongly synergize with H2O2. Moreover, anti-STAT3 antibodies specifically precipitated a protein of 92 kDa that becomes phosphorylated on tyrosine upon lymphocyte treatment with H2O2. Phenylarsine oxide, a tyrosine phosphatase inhibitor, and genistein, a tyrosine kinase inhibitor, cooperated and cancelled, respectively, the H2O2-promoted STAT3 nuclear translocation. Evidence is also presented, using Fe2+/Cu2+ions, that ⋅OH generated from H2O2through Fenton reactions could be a candidate oxygen reactive species to directly activate STAT3. Present data suggest that H2O2 and vanadate are likely to inhibit the activity of intracellular tyrosine phosphatase(s), leading to enhanced STAT3 tyrosine phosphorylation and hence its translocation to the nucleus. These results demonstrate that the DNA binding activity of STAT3 can be modulated by oxidizing agents and provide a framework to understand the effects of oxidative stress on the JAK-STAT signaling pathway.

Pneumocystis jirovecii in General Population

P. jirovecii colonization can be frequently detected in immunocompetent adults, which suggests that the general population could be a source of this infection.

Characterization of Calcineurin in Human Neutrophils INHIBITORY EFFECT OF HYDROGEN PEROXIDE ON ITS ENZYME ACTIVITY AND ON NF-κB DNA BINDING

We describe here a specific calcineurin activity in neutrophil lysates, which is dependent on Ca2+, inhibited by trifluoroperazine, and insensitive to okadaic acid. Immunoblotting experiments using a specific antiserum recognized both the A and B chains of calcineurin. Neutrophils treated with cyclosporin A or FK 506 showed a dose-dependent inhibition of calcineurin activity. The effect of oxidant compounds on calcineurin activity was also investigated. Neutrophils treated with hydrogen peroxide (H2O2), where catalase was inhibited with aminotriazole, exhibited a specific inhibition of calcineurin activity. However, the addition of reducing agents to neutrophil extracts partially reversed the inhibition caused by H2O2. A similar inhibitory effect of H2O2 on calcineurin activity was observed to occur in isolated lymphocytes. This is the first demonstration that redox agents modulate calcineurin activity in a cellular system. In addition, electrophoretic mobility shift assays revealed that lipopolysaccharide-induced activation of NF-κB in human neutrophils is inhibited by cell pretreatment with H2O2 in a dose-dependent manner. These data indicate that calcineurin activity regulates the functional activity of lipopolysaccharide-induced NF-κB/Rel proteins in human neutrophils. These data indicate a role of peroxides in the modulation of calcineurin activity and that the H2O2-dependent NF-κB inactivation in neutrophils occurs in concert with inhibition of calcineurin.

Human neutrophils synthesize IL-8 in an IgE-mediated activation

It has been demonstrated that neutrophils are responsible for the release of large amounts of the inflammatory chemokine interleukin‐8 (IL‐8), associated with inflammation. To further define the mechanisms implicated, we have analyzed the response of human neutrophils from allergic patients to specific antigens or challenge with anti‐immunoglobulin (Ig)E antibodies. Neutrophils showed a dose‐ and time‐dependent production of IL‐8. The release of the cytokine was parallel to expression of IL‐8 mRNA analyzed by the polymerase chain reaction. This expression was transient—it occurred after 3 h of anti‐IgE treatment and was maintained for 18 h. Trifluoperazine, EGTA, reduced nicotinamide adenine dinucleotide phosphate‐oxidase inhibitors, and reactive oxygen species (ROS) scavengers inhibited IL‐8 production, indicating a critical dependence of calcium and oxidative stress. Moreover, an inhibitory effect of cyclosporin A, an immunosuppressor that inhibits calcineurin activity, on IL‐8 release and IL‐8 mRNA expression was observed. This is the first evidence of the involvement of ROS and calcium/calcineurin in IgE‐dependent IL‐8 production. These findings open new perspectives into the functional role of neutrophils in IgE‐associated diseases.

Effect of thimerosal and other sulfhydryl reagents on calcium permeability in thymus lymphocytes

We have studied the effects of thimerosal, a mercurial compound extensively used as a preservative, as well as other sulfhydryl reagents (e.g. p-hydroxymercurybenzoate, hydrogen peroxide, bromophenacyl bromide, and mercuric chloride) on Ca2+ homeostasis and the redox status of sulfhydryl groups in thymus lymphocytes. They all induced an increase in [Ca2+]i which was blocked with dithiothreitol, suggesting that they act via the oxidation or blockade of sulfhydryl groups. [Ca2+]i increase could be directly related to the effect of the different reagents on cellular protein sulfhydryl content. Experiments with ethidium bromide indicate that the observed rise in [Ca2+]i was not due to a non-specific increase in membrane permeability. Thimerosal differs from the other agents studied in its oxidative properties, which is probably linked to the production of a potent reductor molecule, thiosalicylic acid, which may modulate its oxidative capacity.

Modulation of phorbol ester-induced respiratory burst by vanadate, genistein, and phenylarsine oxide in mouse macrophages

The effect of the inhibitors of tyrosine phosphatase (vanadate and phenylarsine oxide) and of an inhibitor of tyrosine kinase (genistein) on O2.- production in mouse peritoneal macrophages was examined. Vanadate and phenylarsine oxide produced a dose-dependent inhibition of phorbol myristate acetate (PMA)-induced O2.- production, whereas genistein potentiated O2.- production triggered by phorbol ester. Vanadate had no effect on the respiratory burst in human neutrophils challenged with fMLP, in agreement with previously published data on human intact neutrophils. It did not alter reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase activity in membrane preparations of mouse peritoneal macrophages. These data suggest that the phosphorylation of protein(s) in tyrosine residues blocked the PMA-dependent respiratory burst in mouse macrophages.

Activation of Peritoneal Macrophages During the Prediabetic Phase in Low-Dose Streptozotocin-Treated Mice

Glucose metabolism and the production of O2 − and NO2 − have been studied in peritoneal macrophages from mice injected with 5 subdiabetogenic doses of streptozotocin. On day 12 after beginning of the treatment, peritoneal macrophages produced significantly higher amounts of lactate than macrophages from control mice. In addition, NO2 − release and phorbol ester‐induced O2 − production were significantly augmented in macrophages from streptozotocin‐treated mice. γ‐Interferon induced in a dose‐dependent manner the activity of NO synthase only in macrophages from streptozotocin‐treated mice. These data show for the first time that peritoneal macrophages from streptozotocin‐treated mice are activated and produce effector molecules such as O2 − and NO which could participate in the destruction of pancreatic islets.

Streptozotocin-induced diabetes increases Fructose 2, 6-biphosfhate levels and glucose metabolism in thymus lymphocytes

Acute effect of streptozotocin-induced diabetes on several parameters of glucose metabolism was investigated in thymus lymphocytes (thymocytes). The cells from diabetics rats accumulated in vitro about 2-fold more fructose 2,6-bisphosphate (Fru-2, 6-P2) in the presence of increasing glucose concentration than cells from normal rats. An increased production of lactate was also observed. Phosphofructokinase-1 (PFK-1) and phosphofructokinase-2 (PFK-2) activities were enhanced in cells from diabetic rats compared with those from normal rats. [U-14C]glucose incorporation into glycogen was also increased in cells from diabetic rats and the 14CO2 liberation was lesser than in cells from normal animals. From these data it may be concluded that the response of thymocytes to streptozotocin-induced diabetes is similar to that observed in other extrahepatic tissues.

Anti-CD69 antibodies enhance phorbol-dependent glucose metabolism and Ca2 levels in human thymocytes. Antagonist effect of cyclosporin A

The human activation antigen CD69 is an early inducible surface glycoprotein acquired by T cells in the thymus at the stage of positive selection and during activation of mature lymphoid cells both in vivo and in vitro. We have studied the regulatory influence of CD69 activation pathway on the glycolytic process and transduction signals of thymocytes. Treatment of human thymocytes with different anti‐CD69 monoclonal antibodies (mAbs), in the presence of submitogenic doses of phorbol ester, produced an enhanced release of lactate without significant alterations in Fru 2,6‐P2 levels or phosphofructokinase‐2 (PFK‐2) and pyruvate kinase activities. A small increase in phosphofructokinase‐1 (PFK‐1) activity was also detected. Furthemore, anti‐CD69 mAb increased the glucose detritiation from [2‐3H] and [3‐3H]glucose, thus indicating an enhanced flux through hexokinase and PFK‐1 steps. In addition, de novo synthesis of diacylglycerol and intracellular Ca2+ levels increased after anti‐CD69 mAb treatment. The stimulatory effects of anti‐CD69 mAb on both glycolysis and Ca2+ levels were inhibited by cyclosporin A. Because CD69 molecules are present in certain subset populations of immature thymocytes, the ability of anti‐CD69 mAb to stimulate the glycolysis, the synthesis of diacylglycerol and the intracellular Ca2+ levels suggest that the activation signals delivered through CD69 molecules could play a role in the thymus cells maturation. J. Leukoc. Biol 60: 278–284; 1996.

NICOTINAMIDE INHIBITS INDUCIBLE NITRIC OXIDE SYNTHASE ENZYME ACTIVITY IN MACROPHAGES BY ALLOWING NITRIC OXIDE TO INHIBIT ITS OWN FORMATION

Nitric oxide (NO) production by macrophages is mainly regulated by induction of nitric oxide synthase (iNOS) by cytokines and microbial products. Nicotinamide (NIC) inhibits NO production by activated macrophages in a dose dependent manner. NIC also inhibits NOS enzyme activity in extracts from activated macrophages. The inhibition was noncompetitive with L-arginine (Ki 13.37 +/- 4.40 mM, n=3), uncompetitive versus NADPH (Ki 3.06 +/- 0.17 mM, n=3) and tetrahydrobiopterin. Finally, the inhibition by nicotinamide was fully reversed by scavenging NO with hemoglobin. We suggest that NIC acts by allowing NO to inhibit its own formation.