Toshihiro Kobayashi

Fungal metabolite gliotoxin inhibits assembly of the human respiratory burst NADPH oxidase.

ABSTRACT Reactive oxygen species are a critical weapon in the killing of Aspergillus fumigatus by polymorphonuclear leukocytes (PMN), as demonstrated by severe aspergillosis in chronic granulomatous disease. In the present study, A. fumigatus-produced mycotoxins (fumagillin, gliotoxin [GT], and helvolic acid) are examined for their effects on the NADPH oxidase activity in human PMN. Of these mycotoxins, only GT significantly and stoichiometrically inhibits phorbol myristate acetate (PMA)-stimulated O2− generation, while the other two toxins are ineffective. The inhibition is dependent on the disulfide bridge of GT, which interferes with oxidase activation but not catalysis of the activated oxidase. Specifically, GT inhibits PMA-stimulated events: p47phox phosphorylation, its incorporation into the cytoskeleton, and the membrane translocation of p67phox, p47phox, and p40phox, which are crucial steps in the assembly of the active NADPH oxidase. Thus, damage to p47phox phosphorylation is likely a key to inhibiting NADPH oxidase activation. GT does not inhibit the membrane translocation of Rac2. The inhibition of p47phox phosphorylation is due to the defective membrane translocation of protein kinase C (PKC) βII rather than an effect of GT on PKC βII activity, suggesting a failure of PKC βII to associate with the substrate, p47phox, on the membrane. These results suggest that A. fumigatus may confront PMN by inhibiting the assembly of the NADPH oxidase with its hyphal product, GT.

Cerium-based cytochemical method for detection of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity at physiological pH.

We have developed a new cytochemical method for detecting the ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase (K-NPPase) activity of the sodium-potassium-activated adenosine triphosphatase (Na-K ATPase) complex. The incubation medium contains p-nitrophenylphosphate (p-NPP) as substrate, cerium chloride as capture agent, Tricine buffer, MgCl2, and KCl. Tricine buffer protected against the medium turbidity caused by non-enzymatic reaction at pH 7.5. Biochemically, the accumulation of p-nitrophenol and phosphate in the reaction precipitate was proportionally related to the enzyme concentration. Ultracytochemically, the reaction products of the K-NPPase activity were localized as fine and uniform electron-dense deposits in the cytoplasmic side of specialized basolateral plasma membranes of cells of kidney distal convoluted tubules, secretory cells of salt gland, and marginal cells of stria vascularis. This method has the advantage of being useful at physiological pH.

Superoxide production at phagosomal cup/phagosome through βI protein kinase C during FcγR-mediated phagocytosis in microglia

Protein kinase C (PKC) plays a prominent role in immune signaling. To elucidate the signal transduction in a respiratory burst and isoform-specific function of PKC during FcγR-mediated phagocytosis, we used live, digital fluorescence imaging of mouse microglial cells expressing GFP-tagged molecules. βI PKC, εPKC, and diacylglycerol kinase (DGK) β dynamically and transiently accumulated around IgG-opsonized beads (BIgG). Moreover, the accumulation of p47phox, an essential cytosolic component of NADPH oxidase and a substrate for βI PKC, at the phagosomal cup/phagosome was apparent during BIgG ingestion. Superoxide (O2−) production was profoundly inhibited by Gö6976, a cPKC inhibitor, and dramatically increased by the DGK inhibitor, R59949. Ultrastructural analysis revealed that BIgG induced O2− production at the phagosome but not at the intracellular granules. We conclude that activation/accumulation of βI PKC is involved in O2− production, and that O2− production is primarily initiated at the phagosomal cup/phagosome. This study also suggests that DGKβ plays a prominent role in regulation of O2− production during FcγR-mediated phagocytosis.

Evaluation of the process for superoxide production by NADPH oxidase in human neutrophils: evidence for cytoplasmic origin of superoxide

Abstract We present an up-to-date insight into the function of NADPH oxidase in human neutrophils, the signalling pathways involved in activation of this enzyme and the process of association of its components with the cytoskeleton. We also discuss the functional implications of morphological studies revealing localization of the sites of NADPH oxidase activity. An original model of the process of superoxide (O2) production in human neutrophils is shown. Organization of NADPH oxidase is associated with several components. Upon stimulation, tri-phox cytosolic components of NADPH oxidase (p40-phox, p47-phox and p67-phox) bind to actin filaments. This process involves other actin-binding proteins, such as cofilin and coronin. Activated protein kinase C, translocated from the plasma membrane, phosphorylates cytosolic components at a scaffold of cytoskeleton. Subsequently, p40-phox, responsible for maintaining the resting state of NADPH oxidase, is separated from other two cytosolic phox proteins following an attachment of the active form of small GTP-binding protein Rac to p67-phox. Cytosolic duo-phox proteins (p47-phox and p67-phox) conjugate with membrane components (gp91-phox, p22-phox and Rap1a) of NADPH oxidase residing within membranes of intracellular compartments. This chain of events triggers production of O2. Then, oxidant-producing intracellular compartments associate with the plasma membrane. Eventually, intracellularly produced O2 is released to the extracellular environment through the orifice formed by fusion of oxidant-producing compartments with the plasma membrane. Intracellular movement of the oxidant-producing compartments may be regulated by myosin light chain kinase. The review emphasizes that functional assembly of NADPH oxidase and, therefore, generation of O2 is accomplished essentially within the intracellular compartments. Upon neutrophil stimulation, intracellularly generated O2 is transported to the plasma membrane to be released and to ensure host defense against infection.

Fungal Metabolite Gliotoxin Targets Flavocytochrome b558 in the Activation of the Human Neutrophil NADPH Oxidase

ABSTRACT Fungal gliotoxin (GT) is a potent inhibitor of the O2−-generating NADPH oxidase of neutrophils. We reported that GT-treated neutrophils fail to phosphorylate p47phox, a step essential for the enzyme activation, because GT prevents the colocalization of protein kinase C βII with p47phox on the membrane. However, it remains unanswered whether GT directly affects any of NADPH oxidase components. Here, we examine the effect of GT on the NADPH oxidase components in the cell-free activation assay. The O2−-generating ability of membranes obtained from GT-treated neutrophils is 40.0 and 30.6% lower, respectively, than the untreated counterparts when assayed with two distinct electron acceptors, suggesting that flavocytochrome b558 is affected in cells by GT. In contrast, the corresponding cytosol remains competent for activation. Next, GT addition in vitro to the assay consisting of flavocytochrome b558 and cytosolic components (native cytosol or recombinant p67phox, p47phox, and Rac2) causes a striking inhibition (50% inhibitory concentration = 3.3 μM) when done prior to the stimulation with myristic acid. NADPH consumption is also prevented by GT, but the in vitro assembly of p67phox, p47phox, and Rac2 with flavocytochrome b558 is normal. Posterior addition of GT to the activated enzyme is ineffective. The separate treatment of membranes with GT also causes a marked loss of flavocytochrome b558s ability to reconstitute O2− generation, supporting the conclusion at the cellular level. The flavocytochrome b558 heme spectrum of the GT-treated membranes stays, however, unchanged, showing that hemes remain intact. These results suggest that GT directly harms site(s) crucial for electron transport in flavocytochrome b558, which is accessible only before oxidase activation.

Isoform-Specific Membrane Targeting Mechanism of Rac during FcγR-Mediated Phagocytosis: Positive Charge-Dependent and Independent Targeting Mechanism of Rac to the Phagosome

Rac1 and Rac2 are capable of stimulating superoxide production in vitro, but their targeting and functional mechanisms are still unknown. In the present study, we found that Rac1, 2, and 3 all accumulate at the phagosome during FcγR-mediated phagocytosis, and that the order of accumulation (Rac1 > Rac3 > Rac2) depends on the net positive charge in their polybasic (PB) regions (183–188 aa). Although all GFP-tagged prenylated PB regions of Rac isoforms (GFP-Rac(PB)) and GFP-tagged prenylated 6 Ala (GFP-6A) accumulated during phagocytosis, GFP-Rac2(PB) and GFP-6A showed weak accumulation at the phagosome through a linear structure connecting the phagosome and endomembranes. The PB region of Rac1 showed strong phospholipid interaction with PI(3)P, PI(4)P, PI(5)P, PI(3,4,5)P3, and phosphatidic acid, however, that of Rac2 did not. Constitutively active Rac2, GFP-Rac2(Q61L), was predominantly localized at the endomembranes; these endomembranes fused to the phagosome through the linear structure during phagocytosis, and this accumulation mechanism did not depend on positive charge in the PB region. Our conclusion is that Rac1 directly targets to the phagosome using the positively charged PB region and this accumulation mechanism is likely enhanced by the phospholipids. In addition to this mechanism, Rac2 has a positive charge-independent mechanism in which Rac2 initially targets to endomembranes and then these endomembranes fuse to the phagosome.

Cooperation of p40phox with p47phox for Nox2-based NADPH Oxidase Activation during Fcγ Receptor (FcγR)-mediated Phagocytosis MECHANISM FOR ACQUISITION OF p40phox PHOSPHATIDYLINOSITOL 3-PHOSPHATE (PI(3)P) BINDING

Background: p40phox acquires PI(3)P-binding capabilities through arachidonic acid-induced and H2O2-induced conformational changes in phagocytes. Results: In addition to conformational changes induced by H2O2 in the cytoplasm, p40phox can acquire PI(3)P binding following membrane targeting. Conclusion: p40phox has novel mechanisms inducing its conformation changes, apart from p47phox. Significance: This study demonstrates both p40phox and p47phox synchronously function as “carriers” and “adaptors” of Nox2-based NADPH oxidase assembly through their conformation changes. During activation of the phagocyte (Nox2-based) NADPH oxidase, the cytoplasmic Phox complex (p47phox-p67phox-p40phox) translocates and associates with the membrane-spanning flavocytochrome b558. It is unclear where (in cytoplasm or on membranes), when (before or after assembly), and how p40phox acquires its PI(3)P-binding capabilities. We demonstrated that in addition to conformational changes induced by H2O2 in the cytoplasm, p40phox acquires PI(3)P-binding through direct or indirect membrane targeting. We also found that p40phox is essential when p47phox is partially phosphorylated during FcγR-mediated oxidase activation; however, p40phox is less critical when p47phox is adequately phosphorylated, using phosphorylation-mimicking mutants in HEK293Nox2/FcγRIIa and RAW264.7p40/p47KD cells. Moreover, PI binding to p47phox is less important when the autoinhibitory PX-PB1 domain interaction in p40phox is disrupted or when p40phox is targeted to membranes. Furthermore, we suggest that high affinity PI(3)P binding of the p40phox PX domain is critical during its accumulation on phagosomes, even when masked by the PB1 domain in the resting state. Thus, in addition to mechanisms for directly acquiring PI(3)P binding in the cytoplasm by H2O2, p40phox can acquire PI(3)P binding on targeted membranes in a p47phox-dependent manner and functions both as a “carrier” of the cytoplasmic Phox complex to phagosomes and an “adaptor” of oxidase assembly on phagosomes in cooperation with p47phox, using positive feedback mechanisms.

COMBINATION TREATMENT OF HYDROGEN PEROXIDE AND X-RAYS INDUCES APOPTOSIS IN HUMAN PROSTATE CANCER PC-3 CELLS

PURPOSE To study the effect of hydrogen peroxide (H(2)O(2)) on radiation-induced apoptosis in human prostate cancer PC-3 cells. METHODS AND MATERIALS At 4h before the irradiation, PC-3 cells were exposed to 10mM ammonium chloride (NH(4)Cl) concentrations. Subsequently, cells were exposed to 0.1mM H(2)O(2) just before the irradiations, which were administered with 10-MV X-rays at doses of 10 Gy. RESULTS The percentage of apoptotic cells at 48 h after X-irradiation alone, H(2)O(2) alone, and combined X-irradiation and H(2)O(2) was 1.85%, 4.85%, and 28.4%, respectively. With use of combined X-irradiation and H(2)O(2), production of reactive oxygen species (ROS) occurred 4h after the irradiation. This resulted in lysosomal rupturing, mitochondrial fragmentation, and the release of cytochrome c into the cytoplasm from the mitochondria. In contrast, when cells were exposed to NH(4)Cl before the X-irradiation and H(2)O(2) administration, apoptosis was almost completely suppressed, ROS production did not occur, lysosomal rupture and mitochondrial fragmentation were blocked, and cytochrome c was not released. CONCLUSIONS Hydrogen peroxide strongly enhanced lysosome-dependent radiation-induced apoptosis in human prostate cancer PC-3 cells. A combined use of X-rays and H(2)O(2) can also injure the mitochondrial cytoplasmic organelles and lead to the production of ROS that in and of itself might possibly induce apoptosis.

Antioxidant Effects of Protocatechuic Acid, Ferulic Acid, and Caffeic Acid in Human Neutrophils Using a Fluorescent Substance

Los neutrofilos humanos estimulados por forbol-miristato-acetato (PMA), un activador de la proteina quinasa C, producen oxigeno activo por la NADPH oxidasa en las estructuras intracelulares. Hemos anadido diacetato de 2´, 7-dihidro dicloro fluoresceina (H2DCFDA), que emite fluorescencia cuando se oxida con las especies de oxigeno activo, a neutrofilos para producir oxigeno activo, a fin de investigar el efecto antioxidante del acido protocatequico, el acido ferulico y el acido cafeico que pertenecen a polifenoles y se distribuyen ampliamente entre las plantas. Particularmente, nos enfocamos en examinar si estas sustancias capturan y eliminan el oxigeno activo dentro o fuera del citoplasma de neutrofilos y si estas sustancias inhiben la NADPH oxidasa. La microscopia de fluorescencia demostro que las estructuras intracelulares positivas a fluorescencia disminuyeron en los neutrofilos mediante la estimulacion de la PMA y exposicion a un antioxidante. La medicion cuantitativa por citometria de flujo revelo que la intensidad de fluorescencia en los neutrofilos, expuestos al acido protocatequico, el acido ferulico, o el acido cafeico, se redujo un 62,9%, 71,4% y 86,1%, respectivamente, en comparacion con las estimuladas por PMA pero no expuestas a un antioxidante. A juzgar desde la microscopia de fluorescencia y la citometria de flujo, estos antioxidantes no tuvieron efectos sobre la morfologia de los neutrofilos. Por otra parte, la intensidad de fluorescencia del oxigeno activo liberado por los neutrofilos se redujeron un 81,4%, 46,7% y 27,4%, respectivamente. El DPI (difenileno-iodonio), un inhibidor especifico de la NADPH oxidasa, inhibio a la enzima en el 92,1% en los neutrofilos estimulados por PMA. El acido protocatequico, el acido ferulico y el acido cafeico inhiben la enzima en un 36,5%, 54,6% y 27,4%, respectivamente. Estos resultados demuestran que el acido protocatequico, el acido ferulico y acido cafeico capturan y eliminan el oxigeno activo, producido por el PMA estimulado por neutrofilos, intracelular y extracelularmente. Ademas, estos antioxidantes inhiben parcialmente la actividad NADPH oxidasa.

Detection of Oxidant Producing-sites in Glutaraldehyde-fixed Human Neutrophils and Eosinophils Stimulated with Phorbol Myristate Acetate

The aim of the present study was to detect oxidant-producing sites, and to elucidate their dynamic reorganization in human polymorphonuclear leukocytes (PMNs) fixed with glutaraldehyde which preserves cell structure. In biochemical analyses, the detectable O2− generation in unfixed PMNs upon stimulation with phorbol 12-myristate 13-acetate (PMA) in the presence of cytochalasin B was characterized by a lag period of approximately 10 sec followed by O2− production. The maximal rate reached was 3.18±0.07 n mol/min/1×106 cells (mean±S.D.; n=4) after 30 sec of stimulation. PMNs exposed to PMA and cytochalasin B followed by fixation with glutaraldehyde generated O2− without a lag period at a rate of 0.35±0.05 n mol/min/1×106 cells (mean±S.D.) by the addition of NADPH as substrate to the cell suspension. In the cytochemical assays, we employed both cells exposed to PMA and cytochalasin B, and then fixed with glutaraldehyde followed by incubation in the cytochemical reaction medium (pre-fixed cells) and cells incubated in the medium containing PMA and cytochalasin B followed by fixation with glutaraldehyde (post-fixed cells). Oxidant reaction in the pre-fixed cells was detected by the addition of NADPH and FAD to the reaction medium. No oxidant-reaction product was seen in pre-fixed cells stimulated for 10 sec whereas the oxidant reaction was visualized in intracellular compartments of pre-fixed PMNs stimulated for 20 sec. The fact that the pre-fixed PMNs stimulated for 30 sec showed increased numbers of oxidant-producing structures compared to those seen in the pre-fixed cells stimulated for 20 sec, demonstrates that the amount of the reaction product and the number of oxidant-producing intracellular compartments increases between 20 and 30 sec after start of stimulation with PMA. These cytochemical results using the pre-fixed cells coincided with the findings obtained from the biochemical assays in the pre-fixed cells exposed to PMA and cytochalasin B. The oxidant reaction was observed in elongated tubular structures that were arranged in a radial fashion, and were associated with the plasma membrane in the pre-fixed PMNs, whereas post-fixed PMNs exhibited slender spherical or rod-shaped structures of various lengths. The present results indicate that the pre-fixed PMNs can be employed for elucidating the dynamic reorganization of oxidant-producing sites in human PMNs.