ABSTRACT
X-linked chronic granulomatous disease (X-CGD) is an inherited immunodeficiency with absent phagocyte NADPH-oxidase activity caused by defects in the gene-encoding gp91(phox). Here, we evaluated strategies for less intensive conditioning for gene therapy of genetic blood disorders without selective advantage for gene correction, such as might be used in a human X-CGD protocol. We compared submyeloablative with ablative irradiation as conditioning in murine X-CGD, examining engraftment, oxidase activity and vector integration in mice transplanted with marrow transduced with a gamma-retroviral vector for gp91(phox) expression. The frequency of oxidase-positive neutrophils in the donor population was unexpectedly higher in many 300 cGy-conditioned mice compared with lethally irradiated recipients, as was the fraction of vector-marked donor secondary CFU-S12. Vector integration sites in marrow, spleen and secondary CFU-S12 DNA from primary recipients were enriched for cancer-associated genes, including Evi1, and integrations in or near cancer-associated genes were more frequent in marrow and secondary CFU-S12 from 300 cGy-conditioned mice compared with fully ablated mice. These findings support the concept that vector integration can confer a selection bias, and suggest that the intensity of the conditioning regimen may further influence the effects of vector integration on clonal selection in post-transplant engraftment and hematopoiesis.
Subject(s)
Bone Marrow/radiation effects , Gene Transfer Techniques , Genetic Vectors , Granulomatous Disease, Chronic/therapy , Hematopoiesis , Retroviridae/genetics , Transplantation Conditioning/methods , Animals , Female , Granulomatous Disease, Chronic/genetics , Hematopoietic Stem Cell Transplantation , Male , Membrane Glycoproteins/genetics , Mice , Mice, Inbred C57BL , NADPH Oxidase 2 , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Neoplasms/genetics , Neutrophils/metabolism , Stem Cells , Transduction, Genetic , Virus IntegrationABSTRACT
Research in gene therapy involving genome-integrating vectors now often includes analysis of vector integration sites across the genome using methods such as ligation-mediated PCR (LM-PCR) or linear amplification-mediated PCR (LAM-PCR). To help researchers analyze these sites and the functions of nearby genes, we have developed SeqMap (http://seqmap.compbio.iupui.edu/) a secure, web-based comprehensive vector integration site management tool that automatically analyzes and annotates large numbers of vector integration sites derived from LM-PCR experiments in human and model organisms upon a common genome database. We believe the use of this resource will enable better reproducibility and understanding of this important data.
Subject(s)
Chromosome Mapping/methods , Genetic Therapy , Integrases/genetics , Internet , Virus Integration/genetics , Animals , Databases, Genetic , Genome , Humans , Polymerase Chain Reaction/methods , Research , Retroviridae/genetics , SoftwareABSTRACT
Phagocyte-derived reactive oxygen species have been implicated in the clearance of malaria infections. We investigated the progression of five different strains of murine malaria in gp91(phox-/-) mice, which lack a functional NADPH oxidase and thus the ability to produce phagocyte-derived reactive oxygen species. We found that the absence of functional NADPH oxidase in the gene knockout mice had no effect on the parasitemia or total parasite burden in mice infected with either resolving (Plasmodium yoelii and Plasmodium chabaudi K562) or fatal (Plasmodium berghei ANKA, Plasmodium berghei K173 and Plasmodium vinckei vinckei) strains of malaria. This lack of effect was apparent in both primary and secondary infections with P. yoelii and P. chabaudi. There was also no difference in the presentation of clinical or pathological signs between the gp91(phox-/-) or wild-type strains of mice infected with malaria. Progression of P. berghei ANKA and P. berghei K173 infections was unchanged in glutathione peroxidase-1 gene knockout mice compared to their wild-type counterparts. The rates of parasitemia progression in gp91(phox-/-) mice and wild-type mice were not significantly different when they were treated with l-N(G)-methylarginine, an inhibitor of nitric oxide synthase. These results suggest that phagocyte-derived reactive oxygen species are not crucial for the clearance of malaria parasites, at least in murine models.
Subject(s)
Immune System/immunology , Malaria/physiopathology , Phagocytes/metabolism , Reactive Oxygen Species/metabolism , Animals , Disease Progression , Glutathione Peroxidase/deficiency , Glutathione Peroxidase/genetics , Glutathione Peroxidase/metabolism , Malaria/immunology , Malaria/metabolism , Mice , Mice, Inbred C57BL , NADPH Oxidases/deficiency , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Nitric Oxide/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Plasmodium berghei/metabolism , omega-N-Methylarginine/pharmacology , Glutathione Peroxidase GPX1ABSTRACT
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.
Subject(s)
Aspergillus fumigatus/pathogenicity , Candida albicans/pathogenicity , Lung Diseases, Fungal/immunology , NADPH Oxidases/physiology , Peroxidase/physiology , Animals , Aspergillosis/immunology , Aspergillosis/microbiology , Candidiasis/immunology , Candidiasis/microbiology , Female , Granulomatous Disease, Chronic/immunology , Granulomatous Disease, Chronic/microbiology , Hypochlorous Acid/metabolism , Lung Diseases, Fungal/microbiology , Mice , Mice, Inbred C57BL , Mice, Knockout , NADPH Oxidases/genetics , Neutrophils/immunology , Neutrophils/metabolism , Peroxidase/geneticsSubject(s)
Malaria/metabolism , Phagocytes/metabolism , Reactive Oxygen Species/metabolism , Animals , Brain/pathology , Brain Edema/etiology , Cerebral Hemorrhage/etiology , Cytoplasmic Granules/enzymology , Female , Hematocrit , Malaria/immunology , Malaria/pathology , Malaria, Cerebral/complications , Malaria, Cerebral/immunology , Malaria, Cerebral/metabolism , Malaria, Cerebral/pathology , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Membrane Glycoproteins/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout , NADPH Oxidase 2 , NADPH Oxidases/blood , NADPH Oxidases/deficiency , NADPH Oxidases/physiology , Parasitemia/immunology , Parasitemia/metabolism , Phagocytes/enzymology , Phagocytosis , Plasmodium bergheiABSTRACT
During the "respiratory burst," the NADPH oxidase complex of phagocytes produces reactive oxygen species that kill bacteria and other invaders (Babior, B. M. (1999) Blood 93, 1464-1476). Electron efflux through NADPH oxidase is electrogenic (Henderson, L. M., Chappell, J. B., and Jones, O. T. G. (1987) Biochem. J. 246, 325-329) and is compensated by H(+) efflux through proton channels that reportedly are contained within the gp91(phox) subunit of NADPH oxidase. To test whether gp91(phox) functions as a proton channel, we studied H(+) currents in granulocytes from X-linked chronic granulomatous disease patients lacking gp91(phox) (X-CGD), the human myelocytic PLB-985 cell line, PLB-985 cells in which gp91(phox) was knocked out by gene targeting (PLB(KO)), and PLB-985 knockout cells re-transfected with gp91(phox) (PLB(91)). H(+) currents in unstimulated PLB(KO) cells had amplitude and gating kinetics similar to PLB(91) cells. Furthermore, stimulation with the phorbol ester phorbol 12-myristate 13-acetate increased H(+) currents to a similar extent in X-CGD, PLB(KO), and PLB(91) cells. Thus, gp91(phox) is not the proton channel in unstimulated phagocytes and does not directly mediate the increase of proton conductance during the respiratory burst. Changes in H(+) channel gating kinetics during NADPH oxidase activity are likely crucial to the activation of H(+) flux during the respiratory burst.
Subject(s)
Membrane Glycoproteins/chemistry , Membrane Glycoproteins/physiology , NADPH Oxidases/chemistry , Phagocytes/metabolism , Protons , Carcinogens , Cell Line , Electrophysiology , Granulomatous Disease, Chronic/genetics , Humans , Hydrogen/metabolism , Ions , Kinetics , Mutation , NADPH Oxidase 2 , Respiratory Burst , Tetradecanoylphorbol Acetate/metabolism , TransgenesABSTRACT
Chronic granulomatous disease (CGD) is an inherited immunodeficiency in which the absence of the phagocyte superoxide-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidase results in recurrent bacterial and fungal infections. A murine model of X-linked CGD (X-CGD) was used to explore variables influencing reconstitution of host defense following bone marrow transplantation and retroviral-mediated gene transfer. The outcomes of experimental infection with Aspergillus fumigatus, Staphylococcus aureus, or Burkholderia cepacia were compared in wild-type, X-CGD mice, and transplanted X-CGD mice that were chimeric for either wild-type neutrophils or neutrophils with partial correction of NADPH oxidase activity after retroviral-mediated gene transfer. Host defense to these pathogens was improved in X-CGD mice even with correction of a limited number of neutrophils. However, intact protection against bacterial pathogens required relatively greater numbers of oxidant-generating phagocytes compared to protection against A fumigatus. The host response also appeared to be influenced by the relative level of cellular NADPH oxidase activity, particularly for A fumigatus. These results may have implications for developing effective approaches for gene therapy of CGD. (Blood. 2001;97:3738-3745)
Subject(s)
Bone Marrow Transplantation/methods , Gene Transfer Techniques , Granulomatous Disease, Chronic/therapy , Animals , Aspergillosis/prevention & control , Aspergillosis/therapy , Burkholderia Infections/prevention & control , Burkholderia Infections/therapy , Disease Models, Animal , Female , Genetic Linkage , Granulomatous Disease, Chronic/complications , Granulomatous Disease, Chronic/enzymology , Immunocompromised Host/immunology , Male , Mice , Mice, Inbred C57BL , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Neutrophils/cytology , Neutrophils/enzymology , Neutrophils/transplantation , Staphylococcal Infections/prevention & control , Staphylococcal Infections/therapy , Transplantation Chimera , X ChromosomeABSTRACT
The phagocyte NADPH-dependent oxidase generates superoxide (O(2)) by reducing molecular oxygen through flavocytochrome b(558) (flavocytochrome b), a heterodimeric oxidoreductase composed of gp91(phox) and p22(phox) subunits. Although each flavocytochrome b molecule contains two heme groups, their precise distribution within the heterodimer is unknown. Among functionally and/or structurally related oxidoreductases, histidines at codons 101, 111, 115, 119, 209, 210, and 222 of gp91(phox) are conserved and potential candidates to ligate heme. We compared biochemical and functional features of normal flavocytochrome b with those in cells expressing gp91(phox) harboring amino acid substitutions at each of these histidines. Surface expression of flavocytochrome b and heterodimer formation were relatively unaffected in cells expressing gp91(phox) H111L, H119L, or H210L. These mutations also had no effect on the flavocytochrome b heme spectrum, although NADPH oxidase activity was decreased in cells expressing gp91(phox) H119L or H210L. In contrast, gp65 was not processed to gp91(phox), heterodimers did not form, and flavocytochrome b was not expressed on the surface of cells expressing gp91(phox) H101L, H115L, H115D, H209C, H209Y, H222L, H222C, or H222R. Similarly, this subset of mutants lacked detectable O(2)-generating activity, and flavocytochrome b purified from these cells contained little or no heme. These findings demonstrate that His(101), His(115), His(209), and His(222) of gp91(phox) are critical for heme binding and biosynthetic maturation of flavocytochrome b.
Subject(s)
Cytochrome b Group/chemistry , Heme/metabolism , Histidine/metabolism , Membrane Glycoproteins/chemistry , NADPH Oxidases , Amino Acid Sequence , Animals , COS Cells , Cytochrome b Group/biosynthesis , Dimerization , Humans , Molecular Sequence Data , NADPH Oxidase 2 , Structure-Activity RelationshipABSTRACT
Flavocytochrome b558 is the membrane component of the phagocyte NADPH oxidase, and is a heterodimer composed of gp91phox and p22phox subunits. Human flavocytochrome b558 is recognized by monoclonal antibody 7D5 at an unidentified extracellular domain, although our previous study suggested it might recognize p22phox. 7D5 has proven useful in rapid screening of individuals for X-linked chronic granulomatous disease by flow-cytometry. Therefore, we re-evaluated the location of the 7D5 epitope using gene-engineered cell lines expressing hybrid flavocytochromes composed of human and murine subunit homologues. The current study demonstrates that the 7D5 recognizes epitope only of primate gp91phox. Flow-cytometric analyses showed that 7D5 consistently bound to cells expressing human gp91phox. In addition, 7D5 immunoprecipitated the approximately 58 kDa unglycosylated gp91phox protein from solubilized membrane fractions of tunicamycin-treated PLB-985 granulocytes, indicating that glycans were not required for 7D5 binding. Transgenic COS7 cells expressing human gp91phox but not p22phox were recognized by 7D5. These results localized the epitope of 7D5 to an extracellular peptide portion of primate gp91phox and indicate that the antibody will be useful for monitoring the efficiency of gene therapy in patients with flavocytochrome b558-deficient chronic granulomatous disease and for elucidating structural characteristics of flavocytochrome b558.
Subject(s)
Antibodies, Monoclonal/immunology , Cytochrome b Group/immunology , Granulocytes/immunology , Membrane Glycoproteins/immunology , NADPH Oxidases/immunology , Primates , 3T3 Cells , Animals , Antibodies, Monoclonal/chemistry , COS Cells , Cattle , Chlorocebus aethiops , Epitopes/analysis , Flow Cytometry , Granulocytes/drug effects , Granulomatous Disease, Chronic/therapy , Humans , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , Mice , NADPH Oxidase 2 , Rabbits , Swine , Transfection , Tunicamycin/pharmacologySubject(s)
Liver/blood supply , Reactive Oxygen Species/metabolism , Reperfusion Injury/prevention & control , Transfection , rac1 GTP-Binding Protein/antagonists & inhibitors , Adenoviridae/genetics , Animals , Apoptosis , Enzyme Induction , Lipid Peroxidation , Liver/enzymology , Liver/pathology , Mice , Mice, Inbred C57BL , NF-kappa B/metabolism , Necrosis , Reperfusion Injury/enzymology , Reperfusion Injury/pathology , rac1 GTP-Binding Protein/geneticsABSTRACT
Intracellular reactive oxygen species (ROS) production by activated murine T lymphocytes was investigated by analyzing intracellular dichlorofluorescin (DCFH(2)) oxidation in lymph node cells (LNC). An increase in DCFH(2) oxidation in LNC induced by phorbol myristate acetate (PMA) was detected by flow cytometry. It was confirmed that this increase was present in Thy1(+) LNC. We examined the contribution to intracellular DCFH(2) oxidation of ROS released by leukocytes other than T cells present in the LNC suspension. Superoxide dismutase, catalase, and glutathione/glutathione peroxidase inhibited the PMA-induced increase in intracellular DCFH(2) oxidation. Furthermore, PMA failed to elicit DCFH(2) oxidation in LNC isolated from mice lacking a functional NADPH oxidase (gp91(phox) gene knockout mice), but this response could be restored in these cells by the addition of T cell-depleted LNC from wild-type litter mates. This study highlights the necessity for caution in using the DCFH(2) assay to demonstrate specific intracellular ROS production in heterogeneous cell populations. It also suggests that cells other than T cells in lymph node populations may, through production of ROS, influence the intracellular redox state of T lymphocytes.
Subject(s)
Fluoresceins/metabolism , T-Lymphocytes/metabolism , Animals , Catalase/pharmacology , Glutathione/pharmacology , Glutathione Peroxidase/pharmacology , Lymph Nodes/cytology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , NADPH Oxidases/deficiency , NADPH Oxidases/metabolism , Oxidation-Reduction , Reactive Oxygen Species/metabolism , Superoxide Dismutase/pharmacology , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
Rac2 is a hematopoietic-specific Rho family GTPase implicated as an important constituent of the NADPH oxidase complex and shares 92% amino acid identity with the ubiquitously expressed Rac1. In bone marrow (BM) neutrophils isolated from rac2(-/-) mice generated by gene targeting, we previously reported that PMA-induced superoxide production was reduced by about 4-fold, which was partially corrected in TNF-alpha-primed BM neutrophils and in peritoneal exudate neutrophils. We investigated receptor-mediated activation of the NADPH oxidase in the current study, finding that superoxide production in rac2(-/-) BM and peritoneal exudate neutrophils was normal in response to opsonized zymosan, reduced to 22% of wild type in response to IgG-coated SRBC, and almost absent in response to fMLP. In wild-type murine BM neutrophils, phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, and Akt was induced by PMA or fMLP, which was decreased in rac2(-/-) neutrophils for ERK1/2 and p38. Activation of p38 by either opsonized zymosan or IgG-coated SRBC was similar in wild-type and rac2(-/-) cells. Inhibition of ERK1/2 or p38 activation using either PD98059 or SB203580, respectively, had only a modest effect on fMLP-elicited superoxide production and no effect on the PMA-induced response. These data provide genetic evidence supporting an important role for Rac2 in regulating neutrophil NADPH oxidase activation downstream of chemoattractant and Fcgamma receptors. The effect of Rac2 deficiency on superoxide production is probably exerted through multiple pathways, including those independent of mitogen-activated protein kinase activation.
Subject(s)
NADP/metabolism , Neutrophils/enzymology , Signal Transduction/immunology , rac GTP-Binding Proteins/physiology , Animals , Biomarkers/analysis , Cells, Cultured , Enzyme Activation/immunology , Enzyme Inhibitors/pharmacology , Humans , Intracellular Fluid/enzymology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/metabolism , N-Formylmethionine Leucyl-Phenylalanine/metabolism , Neutrophil Activation , Neutrophils/metabolism , Nitroblue Tetrazolium/analysis , Receptors, Formyl Peptide , Receptors, IgG/physiology , Receptors, Immunologic/physiology , Receptors, Peptide/physiology , Superoxides/metabolism , p38 Mitogen-Activated Protein Kinases , rac GTP-Binding Proteins/biosynthesis , rac1 GTP-Binding Protein/biosynthesis , RAC2 GTP-Binding ProteinABSTRACT
Despite extensive experimental and clinical evidence demonstrating the critical role of flavocytochrome b558 (Cyt b) in the NADPH-dependent oxidase, there is a paucity of direct structural data defining its topology in the phagocyte membrane. Unlike other Cyt b-specific monoclonal antibodies, 7D5 binds exclusively to an extracellular domain, and identification of its epitope should provide novel insight into the membrane topology of Cyt b. To that end, we examined biochemical features of 7D5-Cyt b binding and used the J404 phage display nonapeptide library to identify the bound epitope. 7D5 precipitated only heterodimeric gp91-p22phox and not individual or denatured Cyt b subunits from detergent extracts of human neutrophils and promyelocytic leukemia cells (gp91-PLB). Moreover, 7D5 precipitated precursor gp65-p22phox complexes from detergent extracts of the biosynthetically active gp91-PLB cells, demonstrating that complex carbohydrates were not required for epitope recognition. Epitope mimetics selected from the J404 phage display library by 7D5 demonstrated that (226)RIVRG(230) and (160)IKNP(163) regions of gp91phox were both bound by 7D5. These studies reveal specific information about Cyt b membrane topology and structure, namely that gp91phox residues (226)RIVRG(230) and (160)IKNP(163) are closely juxtaposed on extracytoplasmic domains and that predicted helices containing residues Gly(165)-Ile(190) and Ser(200)-Glu(225) are adjacent to each other in the membrane.
Subject(s)
Bacteriophages/genetics , Cytochrome b Group/chemistry , Epitope Mapping , NADPH Oxidases , Neutrophils/enzymology , Amino Acid Sequence , Antibodies, Monoclonal/immunology , Cytochrome b Group/genetics , Cytochrome b Group/immunology , Dimerization , Enzyme-Linked Immunosorbent Assay , Humans , Precipitin TestsABSTRACT
Platelet-activating factor (PAF) is an early product of the inflammatory environment, influencing development and resolution of inflammation. Its production is greater in neutrophils and macrophages, which predominantly synthesize 1-alkyl sn-2 acetyl glycerophosphocholine (GPC) than in nongranulocytes (B cells and endothelial cells), which lack a respiratory burst and synthesize 1-acyl sn-2 acetyl GPC as their major PAF species. This study investigated whether the respiratory burst was responsible for the quantitative and qualitative differences in sn-2 acetyl GPC species generation by neutrophils and macrophages versus those cells lacking the NADPH oxidase complex. The myeloid cell line PLB-985 (capable of differentiation into neutrophils) was used to test this hypothesis, since these cells had previously been generated with a non-functional respiratory burst (X-CGD PLB-985). Differentiated PLB-985 cells underwent a large respiratory burst in response to PMA (phorbol ester), and smaller respiratory bursts in response to A23187 (calcium ionophore), and the bacterial polypeptide fMLP (receptor mediated activation). Concurrently, treated cells were assessed for production of 1-hexadecyl and 1-palmitoyl sn-2 acetyl GPC species by gas chromatography/mass spectrometry. Neither cell type generated these lipid species in response to PMA, but both cell types generated equal levels of sn-2 acetyl GPC in response to A23187, with five times more 1-hexadecyl than 1-palmitoyl species. Upon fMLP activation, X-CGD PLB-985 cells produced significantly less 1-hexadecyl and 1-palmitoyl sn-2 acetyl GPC in comparison to the wild-type PLB-985 cells. These findings suggest phagocytic oxidant production by NADPH oxidase is not essential for sn-2 acetyl GPC generation, but appears important for optimal production of PAF in response to some stimuli.
Subject(s)
Glycerylphosphorylcholine/analogs & derivatives , N-Formylmethionine Leucyl-Phenylalanine/analogs & derivatives , NADPH Oxidases/metabolism , Platelet Activating Factor/analogs & derivatives , Platelet Activating Factor/metabolism , Calcimycin , Calcium/metabolism , Cell Line , Gas Chromatography-Mass Spectrometry , Glycerylphosphorylcholine/analysis , Glycerylphosphorylcholine/metabolism , Humans , Ionophores , Leukemia, Myeloid , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Phosphatidylcholines , Platelet Activating Factor/analysis , Platelet Activating Factor/drug effects , Receptors, Formyl Peptide , Receptors, Immunologic/drug effects , Receptors, Immunologic/metabolism , Receptors, Peptide/drug effects , Receptors, Peptide/metabolism , Superoxides/metabolism , Tetradecanoylphorbol Acetate/pharmacologyABSTRACT
BACKGROUND: Chronic granulomatous diseases (CGD) are caused by impaired antimicrobial activity in phagocytes, due to the absence or malfunction of the respiratory burst NADPH oxidase. Two-thirds of the patients have mutations in their X-linked CGD gene encoding gp91phox, the largest subunit of the NADPH oxidase. METHODS: Aimed at gene therapy of X-CGD already at the level of resting pluripotent hematopoietic stem cells, we generated an advanced HIV-1-based vector with self-inactivating (SIN2) features containing the therapeutic gp91phox gene. In this vector an internal cytomegalovirus (CMV) promoter exclusively drives transgene expression. The green fluorescent protein (GFP) served as reporter for evaluation of gene transfer and expression in the human myeloid PLB985 X-CGD cell line. RESULTS: The X-CGD cells were efficiently transduced by the VSV-G pseudotyped lentivirus constructs (up to 74% GFP+ cells at 3 days post-transduction). CMV-driven GFP-expression was stable for at least 3 weeks after transduction and persisted after granulocytic differentiation of the target cells. Using the lentivector with the gp91phox transgene, 26% and 48% of the X-CGD cells expressed gp91phox at Days 2 and 20 after co-culture with 293T producer cells, respectively. Upon granulocytic differentiation of the transduced X-CGD cells with dimethylformamide (DMF), up to 63% (mean 49%, n = 7) of the cells were found to be functionally reconstituted with mean levels of superoxide production of 31% (n = 7) compared to wild-type PLB985 cells. CONCLUSION: Lentivirus vectors expressing gp91phox are able to at least partially correct human myeloid X-CGD cells.
Subject(s)
Gene Transfer Techniques , Granulomatous Disease, Chronic/genetics , Granulomatous Disease, Chronic/therapy , Lentivirus/genetics , Membrane Glycoproteins/genetics , Cell Differentiation , Cell Line , Gene Expression , Genes, Reporter , Genetic Vectors , Granulocytes/cytology , Granulocytes/metabolism , Granulomatous Disease, Chronic/enzymology , Green Fluorescent Proteins , HIV-1/genetics , Humans , Luminescent Proteins/genetics , Mutation , NADPH Oxidase 2 , NADPH Oxidases/genetics , Phenotype , Transduction, Genetic , Vesicular stomatitis Indiana virus/geneticsABSTRACT
BACKGROUND & AIMS: Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used agents that have a high incidence of gastrointestinal side effects resulting in significant morbidity and mortality. Leukocytes have been implicated in NSAID-induced injury, but the mechanisms are unclear. We established a murine model of NSAID-induced gastrointestinal damage to assess the roles of candidate gene products in the pathogenesis of this injury. METHODS: Indomethacin-induced gastrointestinal injury was assessed in wild-type and several mutant murine lines. Leukocyte involvement was assessed by neutrophil depletion, impairment of recruitment (resulting from targeted disruption of fucosyltransferase VII [FTVII]), and the absence of mature T and B cells with the use of Rag 2(-/-) mice. Activation and oxygen free radicals were assessed using gp91(phox-/-) mice that exhibit normal leukocyte recruitment but are deficient in myeloid cell activation and oxygen free radical generation. RESULTS: Impairment of leukocyte recruitment (FTVII(/-)) and neutrophil depletion resulted in more than a 50% reduction in NSAID-induced injury. However, mice deficient in mature T and B cells had NSAID-induced damage comparable to control mice. Leukocyte activation was required for NSAID-induced damage because the gp91(phox-/-) mice were less susceptible to NSAID injury than wild-type mice. CONCLUSIONS: In this murine model system, FTVII-dependent leukocyte recruitment, leukocyte activation via gp91(phox), and neutrophils are required for NSAID-induced gastrointestinal injury, whereas T and B cells are not essential.
Subject(s)
Anti-Inflammatory Agents, Non-Steroidal , Gastrointestinal Diseases/chemically induced , Indomethacin , NADPH Oxidases , Animals , Cell Line , DNA-Binding Proteins/deficiency , DNA-Binding Proteins/genetics , Enteritis/chemically induced , Fucosyltransferases/deficiency , Fucosyltransferases/genetics , Fucosyltransferases/physiology , Gastric Mucosa/metabolism , Gastritis/chemically induced , Gastrointestinal Diseases/metabolism , Intestinal Mucosa/metabolism , Intestine, Small , Leukocytes/physiology , Male , Membrane Glycoproteins/deficiency , Membrane Glycoproteins/genetics , Membrane Glycoproteins/physiology , Mice , Mice, Inbred C57BL , Mice, Knockout/genetics , NADPH Oxidase 2 , Neutrophils/physiology , PermeabilityABSTRACT
Rho GTPases control a variety of cellular processes, including actin polymerization, integrin complex formation, cell adhesion, gene transcription, cell cycle progression, and cell proliferation. A patient is described who has recurrent infections and defective neutrophil cellular functions similar to those found in Rac2-deficient mice. Molecular methods were used to clone the expressed Rac2 cDNA from this patient, and a single base pair change (G-->A at nucleotide 169) in the coding sequence was identified. This results in an asparagine for aspartic acid mutation at amino acid 57 (D57N), a residue that is involved in nucleotide binding and is conserved in all mammalian Rho GTPases. The cloned cDNA was then introduced into normal bone marrow cells through retrovirus vectors, and neutrophils expressing this mutant exhibited decreased cell movement and production of superoxide in response to fMLP. The expressed recombinant protein was also analyzed biochemically and exhibited defective binding to GTP. Functional studies demonstrated that the D57N mutant behaves in a dominant-negative fashion at the cellular level. The syndrome of Rac2 dysfunction represents a human condition associated with mutation of a Rho GTPase and is another example of human disease associated with abnormalities of small G protein signaling pathways. (Blood. 2000;96:1646-1654)
Subject(s)
Phagocytes/immunology , rac GTP-Binding Proteins/genetics , 3T3 Cells , Amino Acid Substitution , Animals , Base Sequence , Bone Marrow Transplantation , Cell Movement , DNA Mutational Analysis , DNA, Complementary/chemistry , DNA, Complementary/genetics , Genes, Dominant , Green Fluorescent Proteins , Guanosine Triphosphate/metabolism , Hematopoiesis , Humans , Infant , Leukocytosis/pathology , Leukocytosis/therapy , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Male , Mice , Microscopy, Fluorescence , Mutation , Neutrophils/cytology , Neutrophils/immunology , Neutrophils/metabolism , Phagocytes/cytology , Point Mutation , Protein Binding , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Retroviridae/genetics , Superoxides/metabolism , rac GTP-Binding Proteins/metabolism , rac1 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/metabolism , RAC2 GTP-Binding ProteinABSTRACT
Chronic granulomatous disease (CGD) is a primary immunodeficiency disorder which results from absence or malfunction of the respiratory burst oxidase normally expressed in neutrophils and other phagocytic leukocytes. Two-thirds of the patients are males hemizygous for mutations in the X-linked gene coding for gp91-phox. As a therapeutic approach towards the X-linked form of CGD bicistronic retroviral vectors containing the gp91-phox gene and a selectable marker gene were constructed. The ability of these vectors to restore NADPH oxidase activity was tested in a human myeloid leukemic cell line that is defective in superoxide production, as well as in primary CD34+ cells obtained from X-CGD patients. Under optimal conditions 80% of the CD34+ cells derived from bone marrow of one X-CGD patient were transduced. The level of superoxide production, in phagocytes derived from transduced cells was 68.9% of normal levels. Considering that low levels of superoxide generating activity are sufficient for normal host defense, the present experiments provide the basis for the development of a gene replacement therapy for the X-linked form of CGD.
Subject(s)
Genetic Therapy/methods , Granulomatous Disease, Chronic/therapy , Animals , Antigens, CD34/metabolism , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , Bone Marrow Cells/virology , Bone Marrow Transplantation , Cell Line , DNA, Complementary/genetics , Gene Expression , Genetic Vectors , Granulomatous Disease, Chronic/genetics , Granulomatous Disease, Chronic/metabolism , Humans , Male , Membrane Glycoproteins/genetics , Mice , NADPH Oxidase 2 , NADPH Oxidases/genetics , Respiratory Burst , Retroviridae/genetics , Superoxides/metabolism , Transduction, Genetic , X Chromosome/geneticsABSTRACT
Mast cells generated from Rac2-deficient (-/-) mice demonstrated defective actin-based functions, including adhesion, migration, and degranulation. Rac2(-/-) mast cells generated lower numbers and less mast cell colonies in response to growth factors and were deficient in vivo. Rac2(-/-) mast cells demonstrated a significant reduction in growth factor-induced survival, which correlated with the lack of activation of Akt and significant changes in the expression of the Bcl-2 family members BAD and Bcl-XL, in spite of a 3-fold induction of Rac1 protein. These results suggest that Rac2 plays a unique role in multiple cellular functions and describe an essential role for Rac2 in growth factor-dependent survival and expression of BAD/Bcl-XL.