Myeloperoxidase deficiency induces MIP-2 production via ERK activation in zymosan-stimulated mouse neutrophils.

Myeloperoxidase (MPO), a major constituent of neutrophils, catalyzes the production of hypochlorous acid (HOCl) from hydrogen peroxide (H2O2) and chloride anion. We have previously reported that MPO-deficient (MPO(-/-)) neutrophils produce greater amount of macrophage inflammatory protein-2 (MIP-2) in vitro than do wild type when stimulated with zymosan. In this study, we investigated the molecular mechanisms governing the up-regulation of MIP-2 production in the mutant neutrophils. Interestingly, we found that zymosan-induced production of MIP-2 was blocked by pre-treatment with U0126, an inhibitor of mitogen-activated protein kinase/extracellular-signal-regulated kinase (ERK), and with BAY11-7082, an inhibitor of nuclear factor (NF)-κB. Western blot analysis indicated that U0126 also inhibited the phosphorylation of p65 subunit of NF-κB (p65), indicating that MIP-2 was produced via the ERK/NF-κB pathway. Intriguingly, we found that ERK1/2, p65, and alpha subunit of inhibitor of κB (IκBα) in the MPO(-/-) neutrophils were phosphorylated more strongly than in the wild type when stimulated with zymosan. Exogenous H2O2 treatment in addition to zymosan stimulation enhanced the phosphorylation of ERK1/2 without affecting the zymosan-induced MIP-2 production. In contrast, exogenous HOCl inhibited the production of MIP-2 as well as IκBα phosphorylation without affecting ERK activity. The zymosan-induced production of MIP-2 in the wild-type neutrophils was enhanced by pre-treatment of the MPO inhibitor 4-aminobenzoic acid hydrazide. Collectively, these results strongly suggest that both lack of HOCl and accumulation of H2O2 due to MPO deficiency contribute to the up-regulation of MIP-2 production in mouse neutrophils stimulated with zymosan.

Relationship of nutrient and residual chlorine concentration in treated wastewater with periphytic algae grown in a stream receiving treated wastewater.

The ratio of the amount of treated wastewater to river water is increasing in urban areas due to the spread of sewage systems. Treated wastewater is also sometimes extensively used to create streams and other water environments in urban areas. Rivers, streams and other water environments provide valuable habitats for all kinds of aquatic species, but the relationship between such aquatic species and the quality of treated wastewater they inhabit is not clearly understood. This study was carried out to clarify the effect of the water quality of treated wastewater such as nutrients and residual chlorine on periphytic algae grown in a stream receiving treated wastewater using laboratory-scale experimental channels. The following results were obtained. (1) When the range of phosphate (PO4-P) concentration was 0.04 to 0.09 mg/L, the higher the PO4-P concentration, the higher the biomass of periphytic algae and the more dominant the Chlorophyceae. (2) When the range of total residual chlorine (TRC) concentration was 0.07 to 5.8 mg/L, the higher the TRC concentration, the lower the biomass of periphytic algae. When the range of TRC concentration was 0.93 to 5.8 mg/L, this tendency was more pronounced.

Earlier onset of neutrophil-mediated inflammation in the ultraviolet-exposed skin of mice deficient in myeloperoxidase and NADPH oxidase.

OBJECTIVE AND DESIGN: This study examined the role of neutrophil-derived reactive oxygen species (ROS) in neutrophil recruitment into ultraviolet B (UVB)-exposed skin of mice. METHODS: Mouse dorsal skin was irradiated with UVB (600 mJ/cm2). Accumulation of neutrophils within the inflammatory sites was observed histochemically. Keratinocyte-derived chemokine (KC) and macrophage inflammatory protein 2 (MIP-2) were quantified, and in vivo chemotaxis of neutrophils toward KC and MIP-2 was examined. RESULTS: UVB exposure of mice deficient in myeloperoxidase (MPO), NADPH oxidase, or both, caused skin neutrophil infiltration peaking at 60, 48, and 48 h, respectively, which was earlier than the 72-h peak in wild-type mice. MIP-2 level was higher in mutant than wild-type mice. Mutant neutrophils produced more MIP-2 in vitro. Neutrophil migration toward a localized source of KC was higher in mutant than wild type mice. NADPH oxidase deficiency had a greater effect on migration than MPO deficiency. CONCLUSIONS: These results suggest that ROS produced by neutrophils regulate expression of MIP-2 and migration of neutrophils toward KC. This may explain the earlier infiltration of mutant neutrophils in response to UVB.

Genetic interaction between DNA polymerase beta and DNA-PKcs in embryogenesis and neurogenesis.

DNA polymerase beta (Polbeta) has been implicated in base excision repair. Polbeta knockout mice exhibit apoptosis in postmitotic neuronal cells and die at birth. Also, mice deficient in nonhomologous end-joining (NHEJ), a major pathway for DNA double-strand break repair, cause massive neuronal apoptosis. Severe combined immunodeficiency (SCID) mice have a mutation in the gene encoding DNA-dependent protein kinase catalytic subunit (DNA-PKcs), the component of NHEJ, and exhibit defective lymphogenesis. To study the interaction between Polbeta and DNA-PKcs, we generated mice doubly deficient in Polbeta and DNA-PKcs. Polbeta(-/-)DNA-PKcs(scid/scid) embryos displayed greater developmental delay, more extensive neuronal apoptosis, and earlier lethality than Polbeta(-/-) and DNA-PKcs(scid/scid) embryos. Furthermore, to study the involvement of p53 in the phenotype, we generated Polbeta(-/-)DNA-PKcs(scid/scid)p53(-/-) triple-mutant mice. The mutants did not exhibit apoptosis but were lethal with defective neurulation at midgestation. These results suggest a genetic interaction between Polbeta and DNA-PKcs in embryogenesis and neurogenesis.

Relative contributions of myeloperoxidase and NADPH-oxidase to the early host defense against pulmonary infections with Candida albicans and Aspergillus fumigatus.

Generation of oxidative products by phagocytic cells is known to be an important host defense mechanism directed toward killing of invading microorganisms. The importance of two major oxidant-producing enzymes, myeloperoxidase (MPO) and NADPH-oxidase, in in vivo fungicidal action was directly compared in genetically engineered mice. Both MPO-deficient (MPO-/-) and NADPH-oxidase-deficient (X-linked chronic granulomatous disease [X-CGD]) mice showed increased susceptibility to pulmonary infections with Candida albicans and Aspergillus fumigatus compared with normal mice, and the X-CGD mice exhibited shorter survivals than MPO-/- mice. This increased mortality of X-CGD mice was associated with a 10- to 100-fold increased outgrowth of the fungi in their organs during the first 6 days. These results suggest that superoxide (O2-) produced by NADPH-oxidase is more important than hypochlorous acid (HOCl) produced by MPO, although both oxidative products obviously contribute to the host defense against pulmonary infection with those fungi. We also observed that MPO-/-/X-CGD double knockout mice showed comparable levels of susceptibility to the X-CGD mice against C. albicans and A. funigatus, indicating that MPO is unable to play a role in host defense in the absence of NADPH-oxidase. This strongly suggests that hydrogen peroxide, the precursor of HOCl, is solely derived from O2- produced by NADPH-oxidase.

Retardation of early-onset PMA-induced apoptosis in mouse neutrophils deficient in myeloperoxidase.

Neutrophil apoptosis is a mechanism involved in the resolution of inflammation. To explore the role of hypochlorous acid (HOCl) produced by neutrophils while they are undergoing apoptosis, we compared the rates of apoptosis in neutrophils isolated from normal mice and from myeloperoxidase (MPO)-deficient mice, which are unable to generate HOCl. Apoptosis in MPO-deficient neutrophils stimulated by phorbol myristate acetate (PMA) was significantly slower than in normal neutrophils during 3 h of incubation. Exposure of normal neutrophils to H(2)O(2) together with PMA resulted in a dramatic acceleration of apoptosis, and almost all of the cells revealed apoptotic morphology at 1 h. This acceleration was inhibited by cytochrome c, a superoxide scavenger. Conversely, in MPO-deficient neutrophils activated with PMA and H(2)O(2), little acceleration was observed before 1 h, although it gradually increased thereafter. This retardation was almost completely reversed when MPO or HOCl was exogenously added. These results suggest that coexistence of HOCl and superoxide accelerates the early onset of neutrophil apoptosis.

DNA polymerase beta is not essential for the formation of palindromic (P) region of T cell receptor gene.

Formation of palindromic (P) region at the variable (V)-diversity (D)-joining (J) junction in DNA polymerase beta (pol-beta) deficient mice were investigated by sequencing of reverse transcriptase-polymerase chain reaction (RT-PCR) products of mRNAs encoding the beta chain of T cell receptor (TCR). Total 42 and 43 cDNA clones encoding V(beta8)-D(beta)-J(beta)-C(beta) from E18.5 embryonic thymocytes of pol-beta gene knocked-out and wild type control mouse, respectively, were sequenced. Among them five and six clones from pol-beta knocked-out and wild type, respectively, have P insertions of two nucleotides. This result unequivocally indicates that pol-beta, which is one of the repair-type DNA polymerases most abundantly expressed in thymus and spleen, is not essential for the formation of P region.

Decreased topoisomerase IIalpha expression confers increased resistance to ICRF-193 as well as VP-16 in mouse embryonic stem cells.

To elucidate the relationship between topoisomerase (topo) II expression and sensitivity to anti-topo II drugs in mammalian cells, we generated mouse embryonic stem cell mutants heterozygous for the topo IIalpha gene by gene targeting. The level of topo IIalpha in the heterozygous cells reduced to one-half of that found in wild-type cells, while topo IIbeta levels were similar in both cell types. Importantly, the heterozygous cells exhibited an increased resistance to ICRF-193 as well as VP-16, suggesting that ICRF-193, like VP-16, exerts its cytotoxicity through converting topo II to a poison.

Contribution of myeloperoxidase to coronary artery vasculitis associated with MPO-ANCA production.

The role of myeloperoxidase (MPO) in the pathogenesis of vasculitis associated with MPO-specific anti-neutrophil cytoplasmic autoantibody (MPO-ANCA) was examined in a murine animal model. Coronary artery vasculitis was induced in C57BL/6 mice with and without endogenous MPO by intraperitoneal injection of Candida albicans-derived substances (CADS). The corresponding levels of MPO-ANCA in sera of mice with and without vasculitis were measured and compared in both wild-type and MPO-deficient animals. The MPO-ANCA titers in sera were significantly higher in mice with vasculitis than in vasculitis-negative mice, indicating that MPO-ANCA correlated with vasculitis formation. However, the increase of MPO-ANCA titers observed in sera of wild C57BL/6 mice were strongly suppressed in MPO-deficient C57BL/6 mice, accompanied with prevention of vasculitis formation. These results show that MPO acted as an antigen for MPO-ANCA production by CADS and was followed by the vasculitis formation. Vasculitis did develop in a few MPO-deficient mice, though the incidence of vasculitis was much lower in MPO-deficient mice than in C57BL/6 mice.

Differential host susceptibility to pulmonary infections with bacteria and fungi in mice deficient in myeloperoxidase.

Myeloperoxidase (MPO), which is located within neutrophils capable of producing hypochlorous acid, is active in vitro against bacteria and fungi. However, MPO-deficient persons are usually healthy. To define the in vivo contribution of MPO to early host defense against pulmonary infections, MPO-deficient and control mice were intranasally infected with various fungi and bacteria, and the number of residual microorganisms in lungs was compared 48 h later. MPO-deficient mice showed severely reduced cytotoxicity to Candida albicans, Candida tropicalis, Trichosporon asahii, and Pseudomonas aeruginosa. However, the mutant mice showed a slight but significantly delayed clearance of Aspergillus fumigatus and Klebsiella pneumoniae and had comparable levels of resistance to the wild type against Candida glabrata, Cryptococcus neoformans, Staphylococcus aureus, and Streptococcus pneumoniae. These results suggest that the MPO-dependent oxidative system is important for host defense against fungi and bacteria, although the effect varies by pathogen species.

Role of myeloperoxidase in the neutrophil-induced oxidation of low density lipoprotein as studied by myeloperoxidase-knockout mouse.

Low density lipoprotein was oxidized by neutrophils derived from either C57BL/6 mice or myeloperoxidase (MPO)-knockout mice. The generation of superoxide from neutrophils of MPO-knockout mice was about 70% of that from wild-type mice. The extent of the oxidation of human low density lipoprotein (LDL) by phorbol myristate acetate (PMA)-activated neutrophils of wild-type and MPO-knockout mice was assessed by measuring consumption of a-tocopherol and formation of phosphatidylcholine hydroperoxide (PCOOH) and cholesteryl ester hydroperoxide (CEOOH). Little consumption of a-tocopherol was observed in both oxidations. It was found, however, that lipid hydroperoxides were accumulated with time in both oxidations and that the rates of formation of PCOOH and CEOOH in the oxidation by MPO-knockout neutrophils were about 66 and 44% of those by wild-type neutrophils, respectively. The lipid peroxidation was completely inhibited by adding superoxide dismutase (SOD) in both cases. The addition of L-tyrosine and SOD enhanced lipid peroxidation of LDL induced by wild-type neutrophils but not by MPO-knockout ones. These results suggest that, regardless of their MPO activity, neutrophils induce lipid peroxidation of LDL by a superoxide-dependent pathway, and that MPO-catalyzed lipid peroxidation is enhanced by the presence of an appropriate amount of free tyrosine and further enhanced by SOD.

Neonatal lethality with abnormal neurogenesis in mice deficient in DNA polymerase beta.

DNA polymerase beta (Polbeta) has been implicated in base excision repair in mammalian cells. However, the physiological significance of this enzyme in the body remains unclear. Here, we demonstrate that mice carrying a targeted disruption of the Polbeta gene showed growth retardation and died of a respiratory failure immediately after the birth. Histological examination of the embryos revealed defective neurogenesis characterized by apoptotic cell death in the developing central and peripheral nervous systems. Extensive cell death occurred in newly generated post-mitotic neuronal cells and was closely associated with the period between onset and cessation of neurogenesis. These findings indicate that Polbeta plays an essential role in neural development.

Severe impairment in early host defense against Candida albicans in mice deficient in myeloperoxidase.

Myeloperoxidase (MPO) catalyzes the reaction of hydrogen peroxide with chloride ion to produce hypochlorous acid (HOCl), which is used for microbial killing by phagocytic cells. Despite the important role of MPO in host defense, however, MPO deficiency is relatively common in humans, and most of these individuals are in good health. To define the in vivo role of MPO, we have generated by gene targeting mice having no MPO activity in their neutrophils and monocytes. The mice without MPO developed normally, were fertile, and showed normal clearance of intraperitoneal Staphylococcus aureus. However, they showed increased susceptibility to pneumonia and death following intratracheal infection with Candida albicans. Furthermore, the lack of MPO significantly enhanced the dissemination of intraperitoneally injected C. albicans into various organs during the first 7 days. Thus, MPO is important for early host defense against fungal infection, and the inability to generate HOCl cannot be compensated for by other oxygen-dependent systems in vivo in mice. The mutant mice serve as a model for studying pulmonary and systemic candidiasis.

Targeted replacement of the mouse apolipoprotein E gene with the common human APOE3 allele enhances diet-induced hypercholesterolemia and atherosclerosis.

Apolipoprotein (apo) E, a constituent of several lipoproteins, is a ligand for the low density lipoprotein receptor, and this interaction is important for maintaining cholesterol and triglyceride homeostasis. We have used a gene replacement strategy to generate mice that express the human apoE3 isoform in place of the mouse protein. The levels of apoE mRNA in various tissues are virtually the same in the human apoE3 homozygous (3/3) mice and their littermates having the wild type mouse allele (+/+). Total cholesterol and triglyceride levels in fasted plasma from the 3/3 mice were not different from those in the +/+ mice, when maintained on a normal (low fat) chow diet. We found, however, notable differences in the distribution of plasma lipoproteins and apolipoprotein E between the two groups: beta-migrating lipoproteins and plasma apoB100 levels are decreased in the 3/3 mice, and the apoE distribution is shifted from high density lipoproteins to larger lipoprotein particles. In addition, the fractional catabolic rate of exogenously administered remnant particles without apoE was 6-fold slower in the 3/3 mice compared with the +/+ mice. When the 3/3 and +/+ animals were fed a high fat/high cholesterol diet, the 3/3 animals responded with a dramatic increase (5-fold) in total cholesterol compared with the +/+ mice (1.5-fold), and after 12 weeks on this same diet the 3/3 animals developed significantly (at least 13-fold) larger atherosclerotic plaques in the aortic sinus area than the +/+ animals. Thus the structural differences between human APOE3 and mouse ApoE proteins are sufficient to cause an increased susceptibility to dietary-induced hypercholesterolemia and atherosclerosis in the 3/3 mice.

Antitumor effect of carboplatin combined with radiation on tumors in mice.

The antitumor effect of cis-diammine-1, 1-cyclobutane dicarboxylate platinum(II) (CBDCA, Carboplatin) and radiation on Ehrlich ascites tumors was evaluated in CD-1 mice. A single dose of CBDCA was combined with a single dose of radiation. The antitumor effect was evaluated by tumor volume. Inhibition of tumor growth by the combination of CBDCA and radiation was greater than by CBDCA and radiation alone. The most effective condition was the simultaneous combination of CBDCA with radiation. Further, we examined the radiation influence on the concentration of platinum in tumor tissue. We found that when CBDCA was administered after irradiation, the concentration of platinum in tumor tissue decreased proportionally with time.

DNA topoisomerase II inhibitors enhance random integration of transfected vectors into human chromosomes.

To study the involvement of DNA topoisomerase (topo) II on nonhomologous (illegitimate) recombination, we examined the effect of topo II inhibitors on random integration of exogenous vectors into human chromosomes. We transfected human cell lines PA1, HeLa and EJ-1 with linearized plasmid pSV2neo by electroporation, treated with topo II inhibitors and determined the frequency of Geneticin-resistant (G418r) colonies. We found that three topo II inhibitors, etoposide (VP-16), ICRF-193 and amsacrine (m-AMSA), greatly enhanced the frequency of G418r colonies. These effects were maximally expressed by as little as 12 hrs treatment with the drugs. Similar enhancements were found with different vectors (closed-circular and linear), different cell types, or by different transfection methods (calcium precipitation and lipofection). In contrast, the inhibitor treatments did not affect the transient expression of chloramphenicol acetyltransferase and beta-galactosidase activity following transfection with pSV2CAT and pCH110, respectively. Southern blot analysis revealed that the integration pattern of transfected pSV2neo into PA1 chromosomes was random and not characteristic for each inhibitor. These results suggest that topo II inhibitors directly act at a nonhomologous recombination reaction, promoting the integration process of transfected vectors into human chromosomes. We discuss the enhancement mechanism with a special emphasis on DNA strand breaks induced by the inhibitors.

Effects of DNA topoisomerase inhibitors on nonhomologous and homologous recombination in mammalian cells.

To study the involvement of DNA topoisomerases in recombination in mammalian cells, we used gene transfer assays to examine the effects of DNA topoisomerase inhibitors on nonhomologous (illegitimate) and homologous recombination. The assays were performed by transfecting adenine phosphoribosyltransferase-deficient (APRT-) CHO cells with plasmids carrying the wild-type or mutant aprt genes and by treating the cells with the inhibitors, followed by subsequent cultivation to select for APRT-positive (APRT+) colonies. Treatments with DNA topoisomerase II inhibitors such as VP-16, VM-26, ICRF-193 resulted in a 3- to 5-fold stimulation of integration of both closed-circular and linearized plasmids carrying the wild-type aprt gene into the recipient genome through nonhomologous recombination. The same treatments also increased 6- to 9-fold and 3-fold the number of APRT+ recombinant colonies that were generated by cotransfecting two closed-circular plasmids with nonoverlapping defective aprt genes and their linearized equivalents, respectively. However, this cotransfection assay involved intrinsically nonhomologous recombination processes; normalization of the frequencies by dividing them with those of the above nonhomologous recombination revealed 2-fold enhancement of homologous recombination events between the circular mutant genes but not between the linear ones. In contrast, DNA topoisomerase I inhibitor, camptothecin, showed no such effect on either recombination. From these results, we discuss the function of DNA topoisomerases on recombination in mammalian cells.

Mice deficient in cystathionine beta-synthase: animal models for mild and severe homocyst(e)inemia.

Studies by various investigators have indicated that elevated levels of plasma homocyst(e)ine are strongly associated with the occurrence of occlusive vascular diseases. With the eventual aim of determining whether or not elevated plasma homocyst(e)ine concentrations are directly causative of cardiovascular diseases, we have generated mice that are moderately and severely homocyst(e)inemic. Homologous recombination in mouse embryonic stem cells was used to inactivate the cystathionine beta-synthase [L-serine hydrolyase (adding homocysteine), EC 4.2.1.22] gene. Homozygous mutants completely lacking cystathionine beta-synthase were born at the expected frequency from matings of heterozygotes, but they suffered from severe growth retardation and a majority of them died within 5 weeks after birth. Histological examination showed that the hepatocytes of homozygotes were enlarged, multinucleated, and filled with microvesicular lipid droplets. Plasma homocyst(e)ine levels of the homozygotes were approximately 40 times normal. These mice, therefore, represent a model for severe homocyst(e)inemia resulting from the complete lack of cystathionine beta-synthase. Heterozygous mutants have approximately 50% reduction in cystathionine beta-synthase mRNA and enzyme activity in the liver and have twice normal plasma homocyst(e)ine levels. Thus, the heterozygous mutants are promising for studying the in vivo role of elevated levels of homocyst(e)ine in the etiology of cardiovascular diseases.

Effect of SV40 and MMTV promoters on intermolecular homologous recombination in rat cells.

The effect of SV40 and MMTV promoters on intermolecular homologous recombination between neomycin (neo) genes carrying deletions proximal and distal to the promoters has been examined in rat XC cells. One deleted neo gene linked to either promoter and the other deleted one without any promoter were co-transfected to cells so that the number of resulting G418-resistant colonies reflected the frequency at which the promoter-linked neo allele had been corrected by homologous recombination. We found that when the the distal deletion was placed under the SV40 promoter, it was corrected over 20-fold more frequently than the proximal. In contrast, when the MMTV promoter replaced the SV40 promoter, the distal deletion was corrected only 2-fold frequently compared with the proximal, and this result did not alter by activating the promoter with a glucocorticoid hormone dexamethasone. These results suggest that the preferential gene correction event observed with the SV40 promoter is attributed not to the transcriptional activity but to some specific sequence of the promoter.

Targeted recombination with single-stranded DNA vectors in mammalian cells.

We studied the ability of single-stranded DNA (ssDNA) to participate in targeted recombination in mammalian cells. A 5' end-deleted adenine phosphoribosyltransferase (aprt) gene was subcloned into M13 vector, and the resulting ssDNA and its double-stranded DNA (dsDNA) were transfected to APRT-Chinese hamster ovary cells with a deleted aprt gene. APRT+ recombinants with the ssDNA was obtained at a frequency of 3 x 10(-7) per survivor, which was almost equal to that with the double-stranded equivalent. Analysis of the genome in recombinant clones produced by ssDNA revealed that 12 of 14 clones resulted from correction of the deletion in the aprt locus. On the other hand, the locus of the remaining 2 was not corrected; instead, the 5' deletion of the vector was corrected by end extension, followed by integration into random sites of the genome. To exclude the possibility that input ssDNA was converted into its duplex form before participating in a recombination reaction, we compared the frequency of extrachromosomal recombination between noncomplementary ssDNAs, and between one ssDNA and one dsDNA, of two phage vectors. The frequency with the ssDNAs was 0.4 x 10(-5), being 10-fold lower than that observed with the ssDNA and the dsDNA, suggesting that as little as 10% of the transfected ssDNA was converted into duplex forms before the recombination event, hence 90% remained unchanged as single-stranded molecules. Nevertheless, the above finding that ssDNA was as efficient as dsDNA in targeted recombination suggests that ssDNA itself is able to participate directly in targeted recombination reactions in mammalian cells.