Chronic Granulomatous Disease Leukocytes Research Articles

Poly Lactic-co-glycolic acid nanoparticles encapsulation enhance the effectiveness of curcumin for rescuing trafficking-defective gp91phox in chronic granulomatous disease leukocytes.

Abstract Mutations in leukocyte NADPH oxidase genes lead to defective reactive oxygen species (ROS) production and cause chronic granulomatous disease (CGD) in humans. Previously we have shown that the sarco/endoplasmic reticulum calcium ATPase (SERCA) pump inhibitor, thapsigargin, rescues the endoplasmic reticulum (ER) retention of the mutant protein in a CGD patient with a gp91phox (H338Y) mutation. Curcumin, a natural constituent of Curcuma longa, is also a SERCA pump inhibitor. However, the clinical effectiveness of curcumin is not satisfactory due to its poor in vivo bioavailability and potential cytotoxicity. In this study, we investigated the effects of organelle-targeting nanoparticles on the delivery of curcumin to leukocytes for the treatment of CGD. First, we investigated the cytotoxicity of curcumin with poly (lactic-co-glycolic acid) (PLGA) nanoparticles encapsulation. Next, we investigated the therapeutic potential of PLGA encapsulated curcumin in mice expressing gp91phox (H338Y) mutant protein. Our results showed that (1) ER-targeting peptide decoration enhanced the localization of PLGA encapsulated curcumin in the ER region; (2) PLGA encapsulated curcumin induced ER calcium release, but the following extracellular calcium entry and ER stress-associated cell death was inhibited; (3) The in vivo test showed that PLGA encapsulated curcumin with ER-targeting peptide decoration enhanced leukocytes gp91phox (H338Y) mutant protein processing and the ROS production. This study showed the therapeutic potential of PLGA encapsulated curcumin for diseases with protein-trafficking defective mutations. Efficient organelle-targeting may promote the efficiency of nanoparticle-based curcumin delivery system.

Thapsigargin and flavin adenine dinucleotide ex vivo treatment rescues trafficking-defective gp91phox in chronic granulomatous disease leukocytes

Mutations in leukocyte NADPH oxidase genes lead to defective respiratory burst in leukocytes and cause chronic granulomatous diseases (CGD) in humans. The most common form of CGD is caused by mutations in the membrane-bound oxidase component gp91phox, which is encoded by the CYBB gene on the X chromosome. We previously reported on a patient with a CYBB mutation (H338Y) that prevents the intracellular trafficking and expression of gp91phox on leukocytes. The capacity of the leukocytes to produce reactive oxygen species (ROS) was rescued by treatment with thapsigargin and flavin adenine dinucleotide (FAD). The increase in ROS production was not due to the increase in cytoplasmic calcium induced by thapsigargin because the treatment of calcium ionophore did not have the same effect. Protein and cellular analyses on leukocytes and cells transfected with GFP-tagged gp91phox mutant showed that treated cells expressed more Endo H-resistant gp91phox protein on the cell surface and are more effective in killing bacteria. Thapsigargin- and FAD-treated CGD leukocytes had enhanced activity in protecting mice from Staphylococcus-induced peritoneal abscess formation in a mouse model of CGD. These results indicate that thapsigargin–FAD ex vivo treatment is effective in rescuing the ROS-producing activity of leukocytes in selected CGD patients.

Enhanced inflammatory responses of chronic granulomatous disease leukocytes involve ROS‐independent activation of NF‐κB

Reactive oxygen species (ROS) generated by the cellular NADPH-oxidase are crucial for phagocytic killing of ingested microbes and have been implicated as signaling molecules in various processes. For example, ROS are thought to be involved in activation of the transcription factor NF-kappaB, central for mediating production of proinflammatory cytokines in response to inflammatory stimuli. Several studies have demonstrated that inhibitors of the NADPH-oxidase interfere with NF-kappaB activation and production of proinflammatory cytokines. Curiously, patients with chronic granulomatous disease (CGD), an immunodeficiency characterized by an inability to produce ROS, are not only predisposed to severe infections, but also frequently develop various inflammatory complications indicative of exaggerated inflammatory responses. Here, we show that human CGD leukocytes display a hyperinflammatory phenotype with increased production of proinflammatory cytokines in response to stimulation with Toll-like receptor agonists. The hyperinflammatory phenotype was also evident in mononuclear cells from CGD mice (gp91(phox) -/-), but not in control cells in the presence of NADPH-oxidase inhibitor diphenyleneiodonium, probably reflecting NADPH-oxidase-independent effects of the inhibitor. Furthermore, we show that the major steps involved in NF-kappaB activation were intact in human CGD cells. These data indicate that ROS were nonessential for activation of NF-kappaB and their production may even attenuate inflammation.

The neutrophil-activating protein (HP-NAP) of Helicobacter pylori is a protective antigen and a major virulence factor.

Helicobacter pylori infection induces the appearance of inflammatory infiltrates, consisting mainly of neutrophils and monocytes, in the human gastric mucosa. A bacterial protein with neutrophil activating activity (HP-NAP) has been previously identified, but its role in infection and immune response is still largely unknown. Here, we show that vaccination of mice with HP-NAP induces protection against H. pylori challenge, and that the majority of infected patients produce antibodies specific for HP-NAP, suggesting an important role of this factor in immunity. We also show that HP-NAP is chemotactic for human leukocytes and that it activates their NADPH oxidase to produce reactive oxygen intermediates, as demonstrated by the translocation of its cytosolic subunits to the plasma membrane, and by the lack of activity on chronic granulomatous disease leukocytes. This stimulating effect is strongly potentiated by tumor necrosis factor alpha and interferon gamma and is mediated by a rapid increase of the cytosolic calcium concentration. The activation of leukocytes induced by HP-NAP is completely inhibited by pertussis toxin, wortmannin, and PP1. On the basis of these results, we conclude that HP-NAP is a virulence factor important for the H. pylori pathogenic effects at the site of infection and a candidate antigen for vaccine development.

Activity of antibiotics in chronic granulomatous disease leukocytes.

Patients with chronic granulomatous disease (CGD) have recurrent infections with catalase-producing organisms, particularly staphylococci, that survive within their leukocytes. To be most effective, antibiotics used to treat infections in CGD patients may need to kill both intracellular and extracellular organisms. We studied the ability of certain antibiotics to penetrate normal and CGD neutrophils and to kill intracellular staphylococci. Trimethoprim and clindamycin were concentrated in normal and in CGD neutrophils; maximum cellular-to-extracellular concentration ratios of clindamycin and of trimethoprim were approximately 30 and approximately 4, respectively. In contrast, penicillin was excluded from normal neutrophils. Clindamycin, trimethoprim/sulfamethoxazole, rifampin, and trimethoprim/rifampin significantly reduced the number of viable intracellular staphylococci in normal and CGD neutrophils. After 24 h of incubation in the presence of these antibiotics, the number of viable intracellular staphylococci in normal and CGD neutrophils was similar. In contrast, dicloxacillin, gentamicin, and cephalothin had no significant effect on the number of intracellular staphylococci in normal or CGD neutrophils.

Utilization of liposomes for correction of the metabolic and bactericidal deficiencies in chronic granulomatous disease.

IgG-coated liposomes containing glucose oxidase (GO) in order to provide a means of generating H2O2 were prepared. Leukocytes from patients with chronic granulomatous disease (CGD) which are lacking a means of generating H2O2, ingested the IgG-coated liposomes and metabolic oxidative deficiencies of glucose-1-14C oxidation and iodination were normalized. Both of these activities have been shown to depend upon the availability of H2O2 within polymorphonuclear leukocytes. Improvement in the capacity to kill Staphylococcus aureus by chronic granulomatous disease leukocytes were observed under similar conditions. Thus, it is possible to restore the oxidative metabolic capacities to CGD leukocytes by the introduction of glucose oxidase containing liposomes to these cells.

Comparison of Myeloperoxidase Activity in Leukocytes from Normal Subjects and Patients with Chronic Granulomatous Disease

A study was undertaken to compare myeloperoxidase (MPO) activity in leukocytes from normal subjects and those with chronic granulomatous disease (CGD) and to eliminate further consideration of MPO as the oxidase responsible for the post-phagocytic respiratory burst and subsequent oxidase microbicidal sequence lacking in leukocytes from patients with CGD. With use of granule fractions isolated from a number of samples, MPO activity in several MPO-mediated biochemical systems (peroxidase assays, protein iodination, and amino acid decarboxylation) was measured. No difference was demonstrated between granule fraction preparations from normal subjects and those with CGD. These data show that MPO activity is normal in CGD leukocytes and are inconsistent with a role for MPO in initiating the post-phagocytic respiratory burst.

Hydrogen Peroxide Production by Leukocytes in Chronic Granulomatous Disease: A Correction

Hydrogen Peroxide Production by Leukocytes in Chronic Granulomatous Disease: A Correction

Allosteric transformation of reduced nicotinamide adenine dinucleotide (phosphate) oxidase induced by phagocytosis in human polymorphonuclear leukocytes.

We used sensitive isotopic and fluorometric assay procedures to investigate reduced nicotinamide adenine dinucleotide (phosphate) [NAD(P)H]oxidation in a particulate fraction derived from normal and chronic granulomatous disease leukocytes. Granules isolated from normal resting cells showed allosteric kinetics with regard to oxidation of either NADH or NADPH, so that no enzyme activity was observed at physiological concentrations of substrate. If the granules were isolated from cells that had previously phagocytized zymosan, normal hyperbolic kinetics were obtained, so that activity could now be observed at low levels of substrate. The activity towards NADPH was always substantially greater than that towards NADH at any given concentration of substrate. This alteration in kinetics with phagocytosis was not observed with the other granule enzymes, acid phosphatase or beta-glucuronidase, and thus appeared to be specific for the reduced pyridine nucleotide oxidase(s). In contrast, granules isolated from cells of patients with chronic granulomatous disease showed allosteric kinetics regardless of whether they were obtained from resting or phagocytizing cells, so that NADPH oxidation was not measurable at physiological concentrations of substrate. This defect in the oxidation of NADPH by granules isolated from phagocytizing chronic granulomatous disease cells was observed over the pH range of 4.0 to 7.0. These data suggest that initiation of the respiratory burst by pahgocytosis normally requires an allosteric transformation in a reduced pyridine nucleotide oxidase, which in turn allows expression of enzymatic activity at physiological concentrations of substrate. The defect in chronic granulomatous disease appears to lie in an inability to achieve this transformation, and the enzyme remains in the inactive, allosteric form.

The Biochemical Basis of Nitroblue Tetrazolium Reduction in Normal Human and Chronic Granulomatous Disease Polymorphonuclear Leukocytes

Normal human polymorphonuclear leukocytes (PMN) placed in anaerobic chambers reaching pO2's of less than 5 mm Hg fail to generate O2-, iodinate ingested particles, and stimulate glucose-1-14C oxidation through the hexose monophosphate shunt. The observation that anaerobic cells are incapable of generating O2- or reducing nitroblue tetrazolium (NBT) to formazan supports the idea that NBT reduction in phagocytizing PMN is due exclusively to oxygen-dependent O2- generating oxidase which is deficient in chronic granulomatous disease leukocytes, despite their hyperphagocytic capacity.

The biochemical basis of nitroblue tetrazolium reduction in normal human and chronic granulomatous disease polymorphonuclear leukocytes

Normal human polymorphonuclear leukocytes (PMN) placed in anaerobic chambers reaching pO2's of less than 5 mm Hg fail to generate O2-, iodinate ingested particles, and stimulate glucose-1–14C oxidation through the hexose monophosphate shunt. The observation that anaerobic cells are incapable of generating O2- or reducing nitroblue tetrazolium (NBT) to formazan supports the idea that NBT reduction in phagocytizing PMN is due exclusively to oxygen-dependent O2- generating oxidase which is deficient in chronic granulomatous disease leukocytes, despite their hyperphagocytic capacity.

The role of superoxide anion generation in phagocytic bactericidal activity. Studies with normal and chronic granulomatous disease leukocytes.

The capacity of human phagocytes to generate superoxide anion (O2-), a free radical of oxygen, and a possible role for this radical or its derivatives in the killing of phagocytized bacteria were explored using leukocytes from normal individuals and patients with chronic granulomatous disease (CGD). Superoxide dismutase, which removes O2-, consistently inhibited phagocytosis-associated nitroblue tetrazolium (NBT) reduction indicating the involvement of O2- in this process. Similarly, superoxide dismutase inhibited the luminescence that occurs with phagocytosis, implicating O2- in this phenomenon, perhaps through its spontaneous dismutation into singlet oxygen. Subcellular fractions from homogenates of both normal and CGD leukocytes generated O2- effectively in the presence of NADH as substrate. However, O2- generation by intact cells during phagocytosis was markedly diminished in nine patients with CGD. Leukocytes from mothers determined to be carriers of X-linked recessive CGD by intermediate phagocytic reduction of NBT elaborated O2- to an intermediate extent, further demonstrating the interrelationship between NBT reduction and O2- generation in phagocytizing cells. Activity of superoxide dismutase, the enzyme responsible for protecting the cell from the damaging effects of O2-, was approximately equal in homogenates of normal and CGD granulocytes. Polyacrylamide electrophoresis separated this activity into a minor band that appeared to be the manganese-containing superoxide dismutase associated with mitochondria and a more concentrated, cyanide-sensitive, cytosol form of the enzyme with electrophoretic mobility that corresponded to that of erythrocyte cuprozinc superoxide dismutase. Superoxide dismutase inhibited the phagocytic killing of Escherichia coli, Staphylococcus aureus, and Streptococcus viridans. A similar inhibitory effect was noted with catalase which removes hydrogen peroxide. Neither enzyme inhibited the ingestion of bacteria. Peroxide and O2- are believed to interact to generate the potent oxidant, hydroxyl radical (.OH). A requirement for .OH in the phagocytic bactericidal event might explain the apparent requirement for both O2- and H2O2 for such activity. In agreement with this possibility, benzoate and mannitol, scavengers of .OH, inhibited phagocytic bactericidal activity. Generation of singlet oxygen from O2- and .OH also might explain these findings. It would seem clear from these and other studies that the granulo cyte elaborates O2- as a concomitant of the respiratory burst that occurs with phagocytosis. To what extent the energy inherent in O2- is translated into microbialdeath through O2- itself, hydrogen peroxide, .OH, singlet oxygen, or some other agent remains to be clearly defined.

Degradation of thyroid hormones by phagocytosing human leukocytes.

Thyroxine (T(4)) and triiodothyronine (T(9)) are rapidly degraded by a purified preparation of myeloperoxidase (MPO) and H(2)O(2) with the formation of iodide and material which remains at the origin on paper chromatography. Deiodination by MPO and H(2)O(2) occurs more readily at pH 7.0 than at pH 5.0 in contrast to iodination by this system which is known to occur more readily at pH 5.0 than at pH 7.0. Degradation is inhibited by azide, cyanide, ascorbic acid, and propylthiouracil. Methimazole stimulates deiodination by MPO and H(2)O(2) but inhibits this reaction when MPO is replaced by lactoperoxidase or horseradish peroxidase.Intact human leukocytes, in the resting state, degrade T(4) and T(3) slowly: degradation, however, is increased markedly during phagocytosis of preopsonized particles. Serum inhibits this reaction. T(3) can be detected as a minor product of T(4) degradation. Proteolytic digestion of the reaction products increases the recovery of monoiodotyrosine. The fixation of iodine in the cytoplasm of leukocytes which contain ingested bacteria was detected radioautographically. Chronic granulomatous disease leukocytes, which are deficient in H(2)O(2) formation, degrade T(4) and T(3) poorly during phagocytosis. MPO-deficient leukocytes degrade the thyroid hormones at a slower rate than do normal leukocytes although considerable degradation is still observed. Azide, cyanide, ascorbic acid, and propylthiouracil which inhibit certain peroxidasecatalyzed reactions inhibit degradation by normal leukocytes; however, inhibition is incomplete. Formation of iodinated origin material is inhibited to a greater degree by azide, cyanide, and propylthiouracil than is deiodination. Methimazole inhibits the formation of iodinated origin material by both normal and MPO-deficient leukocytes. However, deiodination by normal leukocytes is stimulated and that of MPO-deficient leukocytes is unaffected by methimazole. Hypoxia inhibits the degradation of T(4) and T(3) by untreated normal or MPO-deficient leukocytes and by normal leukocytes treated with azide or methimazole. These data suggest that both MPO-dependent and MPO-independent systems are involved in the degradation of T(4) and T(3) by phagocytosing leukocytes. The role of leukocytic degradation of T(4) and T(3) in thyroid hormone economy and in leukocytic microbicidal activity is considered.

Complete deficiency of leukocyte glucose-6-phosphate dehydrogenase with defective bactericidal activity.

A 52 yr old Caucasian female (F. E.) had hemolytic anemia, a leukemoid reaction, and fatal sepsis due to Escherichia coli. Her leukocytes ingested bacteria normally but did not kill catalase positive Staphylococcus aureus, Escherichia coli, and Serratia marcescens. An H(2)O(2)-producing bacterium, Streptococcus faecalis, was killed normally. Granule myeloperoxidase, acid and alkaline phosphatase, and beta glucuronidase activities were normal, and these enzymes shifted normally to the phagocyte vacuole (light and electron microscopy). Intravacuolar reduction of nitroblue tetrazolium did not occur. Moreover, only minimal quantities of H(2)O(2) were generated, and the hexose monophosphate shunt (HMPS) was not stimulated during phagocytosis. These observations suggested the diagnosis of chronic granulomatous disease. However, in contrast to control and chronic granulomatous disease leukocytes, glucose-6-phosphate dehydrogenase activity was completely absent in F. E. leukocytes whereas NADH oxidase and NADPH oxidase activities were both normal. Unlike chronic granulomatous disease, methylene blue did not stimulate the hexose monophosphate shunt in F. E. cells. Thus, F. E. and chronic granulomatous disease leukocytes appear to share certain metabolic and bactericidal defects, but the metabolic basis of the abnormality differs. Chronic granulomatous disease cells lack oxidase activity which produces H(2)O(2); F. E. cells had normal levels of oxidase activity but failed to produce NADPH due to complete glucose-6-phosphate dehydrogenase deficiency. These data indicate that a complete absence of leukocyte glucose-6-phosphate dehydrogenase with defective hexose monophosphate shunt activity is associated with low H(2)O(2) production and inadequate bactericidal activity, and further suggest an important role for NADPH in the production of H(2)O(2) in human granulocytes.

Abnormal bactericidal, metabolic, and lysosomal functions of Chediak-Higashi Syndrome leukocytes.

Phagocytic, antimicrobial, and metabolic functions were studied in leukocytes obtained from three patients with the Chediak-Higashi syndrome (CHS) and compared to normals, individuals, heterozygous for Chediak-Higashi syndrome, and two subjects with chronic granulomatous disease of childhood (CGD). Chediak-Higashi syndrome leukocytes showed normal ingestion of a variety of bacteria, Candida albicans, and polystyrene latex particles. Intracellular destruction was significantly impaired for Staphylococcus aureus, Group D streptococci, and a rough strain of Type II pneumococci over a 2 hr incubation. Killing of Serattia marcescens was consistently delayed at 1 hr whereas that of Escherichia coli and C. albicans appeared normal, unless the incubations were shortened to 20 min. Examination of the rates of killing indicated that the greatest defect occurred in the first 20 min of contact between Chediak-Higashi syndrome cells and bacteria. Separation of Chediak-Higashi syndrome granulocytes from monocytes revealed that the former were most defective in bactericidal activity. After phagocytosis, Chediak-Higashi syndrome granulocytes displayed a normal burst in oxygen consumption and oxidation of glucose-1-(14)C and glucose-6-(14)C and formate-(14)C. Oxidation of glucose-1-(14)C by non-phagocytizing Chediak-Higashi syndrome granulocytes and monocytes averaged 2-3 times normal, whereas glucose-6-(14)C and formate-(14)C oxidation were not significantly increased by resting cells. Iodination of intracellular protein by Chediak-Higashi syndrome leukocytes was significantly increased above normal in both the resting and phagocytizing state. Electron microscopic histochemistry revealed that almost all peroxidase activity was localized to the giant granules in Chediak-Higashi granulocytes, and after bacterial ingestion there was a failure of delivery of peroxidase to many phagosomes. Upon longer incubation more phagosomes acquired peroxidase activity, presumably through a fusion process, although many giant granules remained intact. The contrasting patterns and kinetics of the killing defects and the differing metabolic properties of Chediak-Higashi syndrome and chronic granulomatous disease leukocytes emphasize the pleiomorphic nature of inherited disorders of leukocyte function.

Polymorphonuclear leukocyte myeloperoxidase deficiency in a patient with myelomonocytic leukemia.

SINCE the elucidation of a bactericidal defect in polymorphonuclear leukocytes in chronic granulomatous disease of childhood,1 , 2 other abnormalities of leukocyte function have been reported. Lehr...

Correction of metabolic deficiencies in the leukocytes of patients with chronic granulomatous disease.

Polymorphonuclear leukocytes from patients with chronic granulomatous disease (CGD) exhibit metabolic and bactericidal deficiencies that may be the result of inadequate production of H(2)O(2). A hydrogen peroxide-generating system was, therefore, inserted into CGD leukocytes. This was accomplished by allowing the cells to phagocytize latex spherules coated with glucose oxidase. This produced an amelioration in the known metabolic deficiencies of these cells during phagocytosis: (a) intracellular (catalatic) formate oxidation dependent upon hydrogen peroxide production was enhanced fourfold; and (b) hexose monophosphate shunt activity, which other workers have shown to be at least partially dependent upon the availability of H(2)O(2), was markedly stimulated. These data strengthen the evidence that the fundamental metabolic lesion in CGD cells during phagocytosis is indeed deficient production of hydrogen peroxide, probably, as previously shown, due to diminished oxidase for reduced nicotinamide adenine dinucleotide.

Failure of nitro blue tetrazolium reduction in the phagocytic vacuoles of leukocytes in chronic granulomatous disease.

The leukocytes of patients with chronic granulomatous disease (CGD) may be identified by their failure to reduce Nitro Blue Tetrazolium (NBT) during phagocytosis. This reaction, normally detected in the phagocytic vacuole, is absent or delayed in CGD monocytes and eosinophils as well as in neutrophils, even though sonicates of normal and CGD leukocytes contain equal activities of a cyanide insensitive enzyme system capable of reduction of NBT in the presence of pyridine nucleotide. Enlargement of CGD phagocytic vacuoles appears to be inhibited. Histochemical estimates of the rate of release of alkaline phosphatase are normal in CGD cells. Peroxidase activity is released from CGD cells, but the rate appears to be somewhat slower than normal in some cases. The latter observation may be explained by the increased intensity of the peroxidase stain in resting and phagocytizing CGD cells. The severity of the defect in NBT reduction within the phagocytic vacuoles of the leukocytes of patients and carriers is more variable than was previously appreciated. Some female carriers have profoundly reduced dye reduction and others are nearly indistinguishable from normal. Three brothers with CGD demonstrated significant, albeit delayed, NBT reduction in phagocytic vacuoles during prolonged incubation of their leukocytes. No obvious relationship exists, however, between the rate of reduction of NBT in vacuoles and the clinical severity of the disease.

Defective candidacidal capacity of polymorphonuclear leukocytes in chronic granulomatous disease of childhood

<h2>Summary</h2> Polymorphonuclear leukocytes (PMN) from patients with chronic granulomatous disease of childhood phagocytize <i>C. albicans</i> normally but are unable to kill the intracellular organisms. PMN from normal human adults and newborn infants phagocytize and kill approximately 30 per cent of intracellular organisms in 60 minutes. Sera from control subjects and patients with chronic granulomatous disease are actively opsonic for <i>C. albicans</i>. Heated serum is not opsonic but opsonic capacity can be restored by addition of <i>C. albicans</i> absorbed fresh serum. Both specific heat-stable and nonspecific heat-labile serum factors are necessary for opsonization of <i>C. albicans</i>.

Degranulation of leukocytes in chronic granulomatous disease

Degranulation of leukocytes in chronic granulomatous disease