News and Publications

Regulation of Superoxide Production by Human Neutrophils

A.R. Cross, P.G. Heyworth, B.A. Ellis, D. Noack, L.P. Faust, J.T. Curnutte

Neutrophils are the most numerous phagocytes in the circulating blood and are the first cells to arrive at sites of infection. At these sites, neutrophils ingest invading microorganisms and sequester them within phagocytic vacuoles. Concomitantly, neutrophils activate a highly specialized metabolic pathway, the respiratory burst NADPH oxidase, that results in the production of extremely reactive derivatives of oxygen, such as superoxide, hydrogen peroxide, and hypochlorous acid. These agents have powerful antimicrobial activity and are highly effective in destroying life-threatening pathogens.

In contrast to this beneficial role, when released from the cell, the agents are equally capable of causing serious damage to surrounding normal tissues in inflammatory diseases such as ischemia-reperfusion injury and acute respiratory distress syndrome. It is not surprising, therefore, that neutrophils have an elaborate regulatory mechanism that guards against the inappropriate activation of this pathway but still allows its rapid activation when virulent microbes are present.

One of the 2 major goals of our laboratory is to elucidate the biochemical details of the mechanisms that regulate NADPH oxidase, the phagocytic enzyme that generates superoxide. As we understand this pathway better, it may be possible to develop drugs capable of enhancing its activity in such a way that the antimicrobial efficiency is improved. Conversely, agents that depress the production of oxygen radicals could be used clinically as a new class of antiinflammatory drugs. The other major goal is to understand the molecular basis of chronic granulomatous disease (CGD), in which generation of superoxide by NADPH oxidase is absent or severely diminished, and of related phagocytic disorders in which the production of oxygen radicals is defective.

REGULATION OF NADPH OXIDASE ACTIVITY

NADPH oxidase becomes catalytic when the cytosolic proteins p47^phox and p67^phox become associated with the plasma membrane and form a multimeric complex with the 2 subunits of cytochrome b₅₅₈ (gp91^phox and p22^phox). In addition to heme, the cytochrome probably contains a NADPH-binding site and a flavin group and serves as the electron-carrying component of the system, transferring electrons from NADPH to oxygen. Both p47^phox and p67^phox, together with a third cytosolic protein, p40^phox, exist in a 260-kD complex in the cytosol of resting neutrophils. The 3 proteins translocate to the membrane in a process mediated by interactions between the p47^phox subunit and cytochrome b₅₅₈.

We are actively trying to determine the stoichiometry of the 260-kD cytosolic complex, the proteins with which the components of the complex interact, and the domains in cytochrome b₅₅₈ responsible for the docking of the complex. In addition to the components already described, the activity of NADPH oxidase is also dependent on Rac, a GTP-binding protein of the Ras superfamily. Current studies are directed toward determining the role of Rac2 in regulating the assembly and activation of human neutrophil NADPH oxidase.

In collaboration with J.A. Badwey, Boston Biomedical Research Institute, we are studying the role of protein phosphorylation in NADPH oxidase activation. This study has broadened in scope to include cofilin, a ubiquitous actin-binding protein and a potent depolymerizing agent of filamentous actin. Cofilin is phosphorylated in resting neutrophils and rapidly dephosphorylated when the cells are stimulated to generate superoxide. Using immunofluorescence, we showed that in activated cells cofilin colocalizes with filamentous actin in membrane ruffles and lamellipodia, which are also major sites of NADPH oxidase activity. We have determined that the phosphorylation site on cofilin is Ser3. This amino acid lies within a domain not previously recognized as a target sequence of any known protein kinase. This finding suggests that the phosphorylation state, and hence the activity of cofilin, is regulated by a novel kinase. We are trying to identify this kinase.

Studies are also under way to determine the structure of cytochrome b and the way in which electron transfer between the flavin and heme redox centers is regulated by the cytosolic factors. We showed that the cytochrome contains 2 separate heme centers with closely spaced redox potentials. We also showed that both these centers are contained within the gp91^phox subunit and are not shared between gp91^phox and p22^phox as previously thought. In addition, we found that p47^phox and p67^phox have distinct roles in controlling electron flow from NADPH to oxygen, by regulating both electron flow from NADPH to flavin and from flavin to heme.

These studies suggest multiple ways in which the activity of NADPH oxidase could be artificially controlled. In addition to its capacity to generate superoxide, the oxidase has a mechanism for efficiently transferring protons generated internally from NADPH oxidation to the exterior of the cell to maintain pH homeostasis. We are investigating the mechanisms by which this process can occur and how it is controlled. In collaboration with scientists at The Salk Institute, La Jolla, California, we are investigating the similarities between the neutrophil NADPH oxidase and its homolog in plants, which plays a central role in the resistance response of plants to challenge with pathogens.

MOLECULAR GENETICS OF CHRONIC GRANULOMATOUS DISEASE

Substantial progress has been made both in defining the molecular basis of CGD and in developing improved ways of treating the disease. The CGD phenotype of absent superoxide production can result from genetic lesions affecting any of 4 oxidase components: p47^phox, p67^phox, gp91^phox, or p22^phox. Patients with CGD are highly susceptible to life-threatening microbial infections. More than 170 CGD patients have been assessed in the Scripps General Clinical Research Center. Of these, more than 140 have now been characterized at the molecular genetic level, and a wide variety of mutations have been detected. During the course of these studies, we found that control subjects who do not have CGD have not only a normal copy of the gene for p47^phox but also at least 1 nonfunctional pseudogene. It appears that recombination events between the gene and the pseudogene account for most, if not all, cases of CGD in which p47^phox is affected.

Mutation analysis has also enabled us to provide prenatal diagnosis to a limited number of pregnant female relatives of CGD patients. In conjunction with the Scripps Reference Laboratory, we are developing clinical assays for the diagnosis of CGD and for the detection of specific mutations in patients and families affected by the X-linked (gp91^phox-deficient) form of the disease.

PUBLICATIONS

Adachi, S., Cross, A.R., Babior, B.M., Gottleib, R.A. Bcl-2 and the outer mitochondrial membrane in the inactivation of cytochrome c during Fas-mediated apoptosis. J. Biol. Chem. 272:21878, 1997.

Heyworth, P.G., Curnutte, J.T., Badwey, J.A. Structure and regulation of the NADPH oxidase of phagocytic leukocytes: Insights from chronic granulomatous disease. In: Molecular and Cellular Basis of Inflammation. Serhan, C.N., Ward, P.A. (Eds.). Humana, Totowa, NJ, in press.

Heyworth, P.G., Curnutte, J.T., Rae, J., Noack, D., Cross, A.R. Hematologically important diseases: X-linked chronic granulomatous disease--an update. Blood Cells Mol. Dis. 23:443, 1997.

Heyworth, P.G., Robinson, J.M., Ding, J., Ellis, B.A., Badwey, J.A. Cofilin undergoes rapid dephosphorylation in stimulated neutrophils and translocates to ruffled membranes enriched in products of the NADPH oxidase complex: Evidence for a novel cycle of phosphorylation and dephosphorylation. Histochem. Cell Biol. 108:221, 1997.

Rae, J., Newburger, P.E., Dinauer, M.C., Noack, D., Hopkins, P.J., Kuroto, R., Curnutte, J.T. X-linked chronic granulomatous disease: Mutations in the CYBB gene encoding the gp91^phox component of the respiratory burst oxidase. Am. J. Hum. Genet. 62:1320, 1998.

Roos, D., Curnutte, J.T. Chronic granulomatous disease. In: Primary Immunodeficiency Diseases: A Molecular and Genetic Approach. Ochs, H., Puck, J., Smith, E. (Eds.). Oxford University Press, New York, in press.

Schiff, D.E., Rae, J., Martin, T.R., Davis, B.H., Curnutte, J.T. Increased phagocyte Fc/gammapub/RI expression and improved Fc/gammapub/-receptor-mediated phagocytosis following in vivo recombinant human interferon-/gammapub/ treatment of normal human subjects. Blood 90:3187, 1997.

Yu, L., Quinn, M.T., Cross, A.R., Dinauer, M.C. Gp91^phox is the heme binding subunit of the superoxide-generating NADPH oxidase. Proc. Natl. Acad. Sci. U.S.A. 95:7993, 1998.