Chronic granulomatous disease (CGD), an immunodeficiency with recurrent pyogenic infections and granulomatous inflammation, results from loss of phagocyte superoxide production by recessive mutations in any 1 of 4 genes encoding subunits of the phagocyte NADPH oxidase. These include gp91 phox and p22 phox , which form the membrane-integrated flavocytochrome b, and cytosolic subunits p47 phox and p67 phox . A fifth subunit, p40 phox , plays an important role in phagocytosisinduced superoxide production via a phox homology (PX) domain that binds to phosphatidylinositol 3-phosphate (PtdIns(3)P). We report the first case of autosomal recessive mutations in NCF4, the gene encoding p40 phox , in a boy who presented with granulomatous colitis. His neutrophils showed a substantial defect in intracellular superoxide production during phagocytosis, whereas extracellular release of superoxide elicited by phorbol ester or formyl-methionyl-leucyl-phenylalanine (fMLF) was unaffected. Genetic analysis of NCF4 showed compound heterozygosity for a frameshift mutation with premature stop codon and a missense mutation predicting a R105Q substitution in the PX domain. Parents and a sibling were healthy heterozygous carriers. p40 phox R105Q lacked binding to PtdIns(3)P and failed to reconstitute phagocytosis-induced oxidase activity in p40 phox -deficient granulocytes, with premature loss of p40 phox R105Q from phagosomes. Thus, p40 phox binding to PtdIns(3)P is essential for phagocytosis-induced oxidant production in human neutrophils and its absence can be associated with disease. IntroductionSuperoxide production by the phagocyte NADPH oxidase during the respiratory burst is an essential component of the innate immune response. The active enzyme is assembled from a membrane-bound flavocytochrome b, a heterodimer composed of gp91 phox and p22 phox subunits, and cytosolic regulatory components p47 phox , p67 phox , p40 phox , and the Rac GTPase. 1,2 Genetic defects in the NADPH oxidase cause chronic granulomatous disease (CGD), characterized by absent or markedly reduced enzyme activity, recurrent bacterial and fungal infections, and granulomatous inflammation that can involve multiple organs, including the genitourinary and gastrointestinal tracts. 1,3 Recent retrospective studies report clinical and genetic findings on more than 900 patients. [4][5][6][7] Although there is some ethnic and regional variation, approximately two-thirds of CGD patients have recessive mutations in the X-linked CYBB gene encoding gp91 phox , and the remainder have autosomal recessive defects in CYBA, NCF1, or NCF2, encoding p22 phox , p47 phox , or p67 phox , respectively. Mutations involving p40 phox or Rac have not been reported as causes of CGD. An infant with a dominant-negative mutation in the hematopoietic-specific Rac2 GTPase was reported, with partial oxidase defects, markedly impaired leukocyte migration and adhesion, and a clinical picture that resembled leukocyte adhesion deficiency rather than CGD. 8,9 In the majority of CGD cases, the gen...

Superoxide produced by the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is essential for host defense. Enzyme activation requires translocation of p67phox, p47phox, and Rac-GTP to flavocytochrome b 558 in phagocyte membranes. To examine the regulation of phagocytosis-induced superoxide production, flavocytochrome b558, p47phox, p67phox, and the FcγIIA receptor were expressed from stable transgenes in COS7 cells. The resulting COSphoxFcγR cells produce high levels of superoxide when stimulated with phorbol ester and efficiently ingest immunoglobulin (Ig)G-coated erythrocytes, but phagocytosis did not activate the NADPH oxidase. COS7 cells lack p40phox, whose role in the NADPH oxidase is poorly understood. p40phox contains SH3 and phagocyte oxidase and Bem1p (PB1) domains that can mediate binding to p47phox and p67phox, respectively, along with a PX domain that binds to phosphatidylinositol-3-phosphate (PI(3)P), which is generated in phagosomal membranes. Expression of p40phox was sufficient to activate superoxide production in COSphoxFcγR phagosomes. FcγIIA-stimulated NADPH oxidase activity was abrogated by point mutations in p40phox that disrupt PI(3)P binding, or by simultaneous mutations in the SH3 and PB1 domains. Consistent with an essential role for PI(3)P in regulating the oxidase complex, phagosome NADPH oxidase activation in primary macrophages ingesting IgG-coated beads was inhibited by phosphatidylinositol 3 kinase inhibitors to a much greater extent than phagocytosis itself. Hence, this study identifies a role for p40phox and PI(3)P in coupling FcγR-mediated phagocytosis to activation of the NADPH oxidase.

The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b 558 and cytosolic p67 phox , p47 phox , and p40 phox subunits that undergo membrane translocation upon cellular activation. The function of p40 phox , which binds p67 phox in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40 phox and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40 phox in Fc␥R-induced oxidase activation, we used immunofluorescence and real-time imaging of Fc␥R-induced phagocytosis. YFP-tagged p67 phox and p40 phox translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67 phox and p47 phox accumulation on nascent and internalized phagosomes did not require p40 phox or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40 phox PI3P-binding domain or wortmannin. Translocation of p40 phox to nascent phagosomes required binding to p67 phox but not PI3P, although the loss of PI3P binding reduced p40 phox retention after phagosome internalization. We conclude that p40 phox functions primarily to regulate Fc␥R-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization. (Blood. 2008;112:3867-3877) IntroductionPhagocytic leukocytes are the front-line cellular defense against microbial attack, and are mobilized rapidly to the sites of infection where they ingest and kill opsonized microorganisms. The NADPH oxidase complex plays a central role in this process, as its assembly and activation on phagosomal membranes generate superoxide, the precursor of potent microbicidal oxidants. The importance of this enzyme is demonstrated by genetic defects in the NADPH oxidase complex that cause chronic granulomatous disease (CGD), characterized by recurrent severe and potentially lethal bacterial and fungal infections. 1 The NADPH oxidase includes the membrane-integrated flavocytochrome b, composed of gp91 phox and p22 phox , and the cytosolic components p47 phox , p67 phox , p40 phox , and Rac, a Rho-family GTPase, which translocate to flavocytochrome b upon cellular stimulation to activate superoxide production. [2][3][4] Segregation of regulatory components to the cytosol in resting cells facilitates the temporal and spatial regulation of NADPH oxidase activity. The p67 phox subunit is a Rac-GTP effector 2-4 containing a domain that activates electron transport through the flavocytochrome. 5 In resting cells, p67 phox is associated with p40 phox via complementary PB1 (phagocyte oxidase and Bem1p) motifs present in each protein. 2,[6][7][8] p67 phox is also linked to p47 phox via a high-affinity interaction involving an SH3 domain and a proline-rich region, respectively, in the C-termini of...

Chronic granulomatous disease (CGD) is a primary immunodeficiency syndrome characterized by a greatly increased susceptibility to severe fungal and bacterial infections. CGD results from a failure of the reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase enzyme in the patient's phagocytes to produce superoxide. It is caused by mutations in any of four genes that encode the components of the NADPH oxidase. Investigation of CGD patients has identified the different subunits and the genes encoding them. Study of rare CGD variants has highlighted sequences involved in the structural stability of affected components or has provided valuable insights into their function in the oxidase activation mechanism. Functional and molecular CGD diagnosis tests are discussed in this review. Long-term antibiotic prophylaxis has been essential in fighting infections associated with CGD, but approaches based on hematopoietic stem cell transplantation and gene therapy offer great hope for the near future.