Reconstitution of bactericidal activity in chronic granulomatous disease cells by glucose-oxidase–containing liposomes

Abstract

Chronic granulomatous disease (CGD) is an inherited primary immunodeficiency characterized by phagocytes devoid of a functioning nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. The failure of CGD phagocytes to produce reactive oxygen species (ROS) results in a marked increase in the susceptibility of affected patients to life-threatening bacterial and fungal infections. This study investigated whether loading of CGD phagocytes with glucose oxidase (GO)-containing liposomes (GOLs) could restore cellular production of bactericidal ROS (eg, H 2 O 2 and HOCl) in vitro. Results indicate that GO encapsulated in liposomes enabled NADPH oxidase-deficient phagocytes to use H 2 O 2 for the production of highly bactericidal HOCl. The intracellular colocalization of bacteria and liposomes (or liposome-derived ferritin) was demonstrated by confocal laser microscopy and electron microscopy. After uptake of GOLs (approximately 0.2 U/mL at 1 mM total lipid concentration, size approximately 180 nm), CGD granulocytes produced HOCl levels comparable to those of normal phagocytes. Remarkably, after treatment with GOLs, CGD phagocytes killed Staphylococcus aureus as efficiently as normal granulocytes. Moreover, treated cells retained sufficient motility toward chemotactic stimuli as measured by chemotaxis assay. Side effects were evaluated by measuring the H 2 O 2 concentrations and the production of methemoglobin in whole blood. These studies revealed that H 2 O 2 produced by GOLs was degraded immediately by the antioxidative capacity of whole blood. Elevated methemoglobin levels were observed only after application of extremely high amounts of GOLs (2 U/mL). In summary, the application of negatively charged GOLs might provide a novel effective approach in the treatment of patients with CGD at high risk for lifethreatening infections. IntroductionChronic granulomatous disease (CGD) is a primary immunodeficiency with an incidence of 1:1 000 000 to 1:250 000. 1 It is an inherited disorder (X chromosomal or autosomal recessive) of the phagocyte system. 2 Phagocytes (granulocytes and monocytes) of patients with CGD lack a functional nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and are not able to produce reactive oxygen species (ROS), necessary for microbial killing. 3 The disease is therefore dominated by high susceptibility to a variety of bacteria and fungi, especially those with catalase activity. 4 Therapy and prevention of infections include the life-long administration of antibiotics and antimycotics, and in severe cases, the transfusion of allogeneic granulocytes to provide the patient with functioning phagocytes. 5 In recent years the idea of encapsulating drugs such as antibiotics or cytostatics into liposomal carriers has become more feasible. 6 Phagocytes tend to take up conventional liposomes very easily. 7 This uptake is often a disadvantage in the use of liposomes for many medical applications and clinical trials because of rapid elimination of the encapsulated drug. We developed glucose oxid...

Liver-homing of purified glucose oxidase: A novel in vivo model of physiological hepatic oxidative stress (H2O2)

“…In contrast to other studies [38,[45][46][47][48], our approach uses the A. niger-derived GOX in an unbound form and at well-defined concentrations, allowing to increase and titer steady-state H 2 O 2 concentrations to levels comparable to those sustained by activated neutrophils and Kupffer cells in vivo [19,20]. GOX is rapidly phagocytosed and, after a transient passage via the spleen, eventually and exclusively targeted to the liver sinus with a maximum activity after 16 h. In the liver it is primarily found in Kupffer cells as shown by immunofluorescence.…”

Clearance of Apoptotic Neutrophils Is Diminished in Cord Blood Monocytes and Does Not Lead to Reduced IL-8 Production

“…Neutrophils were isolated from the density gradient centrifugation pellet by hypotonic lysis of the remaining erythrocytes using 0.156 M ammonium chloride (18). Neutrophils were washed twice with PBS (Biochrom) and counted with an ultra plane Neubauer hemocytometer.…”

NOX2 As a Target for Drug Development: Indications, Possible Complications, and Progress

“…The central role of the NOX2 system in host defense is demonstrated by the inherited condition chronic granulomatous disease (CGD) in which phagocytes genetically defective in NOX2 or one of its interacting regulatory subunits fail to kill ingested microorganisms, despite normal phagocytosis, resulting in frequent and chronic infections in affected individuals. Definitive evidence for the microbicidal role of ROS was provided by experiments which showed that exogenous H 2 O 2 could restore the ability of defective neutrophils from CGD neutrophils to kill microorganisms (87). Neutrophils from mice in which NOX2 or one of its regulating subunits is genetically deleted show identical microbicidal defects (119,223).…”