Structural organization of the neutrophil NADPH oxidase ...

Structural organization of the neutrophil NADPH oxidase:phosphorylation and translocation during primingand activation

Forest R. Sheppard,*,† Marguerite R. Kelher,†,‡ Ernest E. Moore,*,† Nathan J. D.McLaughlin,‡,§ Anirban Banerjee,† and Christopher C. Silliman†,‡,§,1

*Department of Surgery, Denver Health Medical Center, Colorado; Departments of †Surgery and §Pediatrics,University of Colorado School of Medicine, Denver; and ‡Bonfils Blood Center, Denver, Colorado

Abstract: The reduced nicotinamide adeninedinucleotide phosphate (NADPH) oxidase is part ofthe microbicidal arsenal used by human polymor-phonuclear neutrophils (PMNs) to eradicate invad-ing pathogens. The production of a superoxideanion (O2

–) into the phagolysosome is the precur-sor for the generation of more potent products,such as hydrogen peroxide and hypochlorite.However, this production of O2

– is dependent ontranslocation of the oxidase subunits, includinggp91phox, p22phox, p47phox, p67phox, p40phox, andRac2 from the cytosol or specific granules to theplasma membrane. In response to an external stim-uli, PMNs change from a resting, nonadhesive stateto a primed, adherent phenotype, which allows formargination from the vasculature into the tissueand chemotaxis to the site of infection upon acti-vation. Depending on the stimuli, primed PMNsdisplay altered structural organization of theNADPH oxidase, in that there is phosphorylationof the oxidase subunits and/or translocation fromthe cytosol to the plasma or granular membrane,but there is not the complete assembly required forO2

– generation. Activation of PMNs is the completeassembly of the membrane-linked and cytosolicNADPH oxidase components on a PMN mem-brane, the plasma or granular membrane. Thisreview will discuss the individual components asso-ciated with the NADPH oxidase complex and thefunction of each of these units in each physiologicstage of the PMN: rested, primed, and activated. J.Leukoc. Biol. 78: 1025–1042; 2005.

Key Words: review � innate immunity � respiratory burst � oxidaseassembly

HISTORICAL BACKGROUND

In 1933, it was observed that phagocytic cells, mainly thepolymorphonuclear neutrophil (PMN), demonstrated markedlyincreased oxygen consumption, or a respiratory burst, duringphagocytosis [1, 2]. This increased oxygen consumption waspostulated to be related to an increased energy demand for the

phagocytic process and was assumed to be mitochondrial innature. However, pretreatment of leukocytes with mitochon-drial poisons, such as cyanide and azide, did not inhibitconsumption, thus demonstrating that the increased oxygenconsumption was not a result of an elevated energy require-ment for phagocytosis [3]. Soon thereafter, investigators estab-lished that this respiratory burst was required for the efficientkilling of bacteria by PMNs [4]. In 1967, the importance ofthese findings was realized by the identification of chronicgranulomatous disease (CGD), an illness defined by the ab-sence of the respiratory burst, poor bactericidal capabilities,and death as a result of overwhelming infections [5]. Con-versely, in the early 1970s the PMN was implicated in thepathogenesis of acute lung injury (ALI), recognizing PMNs aspotentially injurious to the host [6, 7]. These observations haveled to an improved understanding of the vital role PMNs playin host defense and tissue damage—the latter as the result ofproinflammatory activation of the microbicidal response as aresult of injury, ischemia reperfusion, and other underlyingclinical conditions.

OXIDASE ACTIVITY: GENERATION OFTHE SUPEROXIDE ANION (O2

–) ANDDOWNSTREAM BYPRODUCTS

The PMN oxidase accepts an electron from reduced nicotin-amide adenine dinucleotide phosphate (NADPH) at the cyto-solic surface of the plasma membrane and donates it to mo-lecular oxygen on the other side in the phagolysosme or to theimmediate, extracellular environment, generating a O2

–

[8–13]. The generated O2– can subsequently be converted to

other cytotoxic products [14–17], and the majority of O2–

produced is dismutated to hydrogen peroxide (H2O2) [18],mainly through the granular enzyme myeloperoxidase (MPO).The generated H2O2 functions in the following ways: 1) oxi-dizes a variety of aromatic compounds (R-H) by electron trans-fer, yielding substrate radicals [19–21]; 2) oxidizes chloride

1 Correspondence: Bonfils Blood Center, 717 Yosemite St., Denver, CO80230. E-mail: [email protected]

Received April 20, 2005; revised July 15, 2005; accepted July 18, 2005;doi: 10.1189/jlb.0804442

0741-5400/05/0078-1025 © Society for Leukocyte Biology Journal of Leukocyte Biology Volume 78, November 2005 1025

ions to the nonradical oxidant hypochlorous acid, the PMNsmost potent bactericidal product [22]; 3) converts to the highlyreactive hydroxyl radical by the Fenton reaction between theH2O2 and a transition metal catalyst (Fe3

�/Fe2�) [23–25]; 4)

generates singlet oxygen, an additional, albeit minor, byprod-uct of O2

–, which is an extremely energetic form of oxygen,

capable of attacking double bonds [9]; and 5) produces reactivenitrogen species from nitric oxide (NO); however, data con-cerning the ability of PMNs to directly generate the needed NOfor this reaction are controversial [26].

THE PMN NADPH OXIDASE COMPONENTS

The NADPH oxidase, comprised of membrane [gp91phox (wherephox stands for phagocyte oxidase), p22phox, and the smallG-protein Rap1A] and cytosolic (p47phox, p67phox, p40phox, thesmall G-proteins Rac2 and Cdc42, and the newly identifiedp29 peroxiredoxin) components, has been described and char-acterized in the PMN, mainly from studies about CGD [27–33].The membrane-bound subunits gp91phox and p22phox togetherform the heterodimeric cytochrome b558. Upon oxidase activa-tion, the cytosolic subunits p47phox, p67phox, and p40phox trans-locate to the plasma membrane and bind with the cytochromeb558 complex [34, 35]. Additionally, the small GTPase proteinsRac2, Cdc42, and Rap1A are involved in the assembly andactivation of the NADPH oxidase [36–38]. A new protein, p29peroxiredoxin, associated with the oxidase proteins (mainlyp67phox) has recently been described [39]. The individualcomponents and their role in the function of the NADPHoxidase, as related to the human PMN, are described in detailbelow and summarized (see Figs. 1–3).

THE MEMBRANE COMPONENTS OFNADPH OXIDASE

Cytochrome b558

Cytochrome b558 is a membrane-bound flavohemoprotein,which functions to transfer electrons supplied by the cytosolicNADPH across the membrane to a phagolysosme or an extra-cellular molecular oxygen [40–42]. This stable heterodimer iscomposed not only of p22phox (�-subunit) and gp91phox (�-subunit) but also a flavin adenine dinucleotide (FAD), whichserves as a NADPH-binding site, and two heme prostheticgroups, one of which selectively binds gp91phox, and the otherbinds gp91phox and p22phox [42–47].

The FAD and heme groups serve as the redox pathway,which enables the transfer of electrons across the membrane.The two heme groups are functionally distinct from each other[48], and there is controversy concerning the role of the hemeprosthetic groups as obligatory intermediates for oxidase ac-tivity [42, 49–52]. On one side, if heme oxidation is anobligatory participant in electron transfer, then the maximalrate of O2

– production would be significantly diminished; fur-thermore, O2

– generation is not inhibited by the heme antag-onists CN–, N3

–, CO, and butyl isonitrile [45, 53–56]. Con-versely, the heme prosthetic groups serve as an intermediate

between the FAD and O2– generation, as the two heme pros-

thetic groups have binding sites to gp91phox and p22phox [42,50, 51, 57–61], affect or are affected by the cytosolic oxidasecomponents upon translocation [42, 50–52, 56, 60, 61], andtherefore, may be important in the assembly of the NADPHcomplex. In a cell-free system, the heme group was found to bereduced by NADPH in the plasma membrane, but it may not beinvolved in the catalytic activity of the oxidase [54, 62, 63].However, despite the controversy regarding the heme subunits,there is a consensus among investigators that the FAD subunitis an electron carrying intermediate in O2

– generation, asoxidase activity is lost when the FAD is removed but restoredwhen added back. Moreover, the oxidase activity can be in-hibited by flavin antagonists (e.g.. deaza-FAD and diphenyleneiodonium) [10, 64, 65]. It is important to understand thefunction and role of these subunits to better understand andregulate the NADPH oxidase and O2

– generation.In resting PMNs, 15% of the cytochrome b558 subunits

gp91phox and p22phox are located in the plasma membrane andthe remaining 85%, within the membrane of the specific gran-ules and secretory vesicles [66–69] and then translocated tothe plasma membrane for oxidase activation [70, 71]. More-over, a defect in either of the subunits results in the completeabsence of the heterodimer from the plasma membrane [52, 72]and also a deficiency of that subunit in the specific granules,resulting in decreased oxidase activity [73–75]. The gp91phox

and p22phox proteins are reviewed briefly below.The mature p22phox (�-subunit) protein binds to gp91phox

(�-subunit) at the membrane, creating a stable cytochrome b558

complex [27, 71]. The p22phox protein contains a proline-richregion on its cytoplasmic tail, which interacts with src homol-ogy region 3 (SH3) domains, including the SH3 domain ofphosphorylated p47phox [76, 77]. Along with gp91phox, p22phox

binding of the cytosolic oxidase components, specificallyp47phox and p67phox, is essential for proper function of therespiratory burst [78–81]. Defects and/or deficiencies inp22phox have been reported in CGD patients [72, 82–86].However, these defects tend to be autosomal instead of X-linked like defects in gp91phox [85, 86]. It is interesting thatthere is a significant decrease (33%) in O2

– production with theTT genotype of the C242T polymorphism and no link to adecrease in immune function; however, an increased risk ofcoronary artery disease is observed [87].

The gp91phox (�-subunit) is post-translationally glycosylatedon three of the potential five N-linked glycosylation sites[88–90], contains five hydrophobic domains on the N-termi-nus, which likely represent membrane-spanning domains [91,92], and has a cytosolic, hydrophilic region at the C-terminus,which contains the heme moieties and interacts with p47phox

[91, 93]. Blocking of the C-terminus inhibits oxidase activitybut not translocation or binding of the cytosolic subunits to theplasma membrane; thus, gp91phox appears essential for oxidasefunction [93]. The role of gp91phox is speculated to be a H�

channel, permitting charge compensation across the mem-brane, coinciding with electron transfer, regulated by the trans-located Rac2 and p67phox [94], and gp91phox may also regulatethe steady-state FAD reduction [95].

Mutations in gp91phox are X-linked and the most commondefect in CGD. These mutations include A57E, E309K,

1026 Journal of Leukocyte Biology Volume 78, November 2005 http://www.jleukbio.org

C537R, P339H, �F215, and �F216 [96–98]. The �F215 and�F216 mutations result in aberrant phenylalanine residues,and affected cells contain no trace of cytochrome b558, sug-gesting that one or both of these phenylalanine residues areessential for the binding of cofactors to the membrane-boundcytochrome [97]; however, there is a normal amount of gp91phox

present in the PMN. The carrier state for a gp91phox defect isalso detrimental, leading to a depressed immune response,colonization with fungi, and resultant granulomatosis [99]. Ingp91phox�/� mice, there is a lack of O2

– production, nochange in p22phox, greater PMN sequestration in the lungs, anda down-regulation in cyclooxygenase 2 (COX-2) [100–104].

Rap1A (Krev 1)

Rap1A (also referred to as Krev1) is a low molecular weight (22kDa), guanosine 5�-triphosphate (GTP)-binding protein, whichis a member of the Ras superfamily and found in large quan-tities in PMNs [37, 105–107]. Its role in the regulation of theoxidase was initially suggested by its copurification and coim-munoprecipitation with cytochrome b558 (1:1 stoichiometry)[37, 38, 91, 108]. Rap1A translocates with cytochrome b558

from the specific granules to the plasma membrane upon cellstimulation, but this interaction is inhibited by cyclic adeno-sine monophosphate (CAMP)-dependent protein kinase A(PKA) phosphorylation [38, 106, 109]. When Rap1A binds tocytochrome b558, the guanosine-5�-O-3-thiotriphosphate (GTP-gamma-s)-bound form of Rap1A binds more tightly to cyto-chrome b558 than the guanosine diphosphate-bound form(GDP) [108]. Phospholipase C (PLC) activation, leading toelevated levels of intracellular-free Ca2� and diacylglycerol(DAG), has been implicated as a mediator of Rap1A activationvia PKC phosphorylation [105, 110, 111]. The role of Rap1A inoxidase regulation is controversial; some reports show thatdepleting the cell of Rap1A decreases oxidase activity [36, 37,105–107], and other reports indicate that there is no effect onthe oxidase [108, 112]. In HL-60 cells, Rap1A was suggestedto act as the final activation switch of the NADPH oxidase byits interaction with the cytochrome b558 [36]. The truncatedform of Rap1A in a cell-free system was functional in NADPHactivation but may not have a direct role in the regulation of theoxidase, as there is no change in expression in patients withCGD [36, 113]. Further work is required to fully define the roleof Rap1A in oxidase activation and the role of protein kinaseisoforms in overall oxidase function.

Nox proteins

Nox proteins, or NADPH oxidase enzymes, are homologs togp91phox (Nox2), which are found mainly in nonphagocyticcells [45, 114, 115]. In humans, there are seven members ofthis family, but Nox1, Nox3, and Nox4 have the most homol-ogous and similar structure to Nox2 [114, 116, 117]. Nox1,along with Nox4, are structurally the most similar to Nox2, arefound in epithelial cells, and colocalize with p22phox for O2

–

generation [117–120], and Nox3 is found primarily in fetaltissues and is possibly related to development [114, 116].Recently, however, Nox proteins have been shown to be re-sponsible for reactive oxygen species (ROS) generation innonphagocytic cells [114, 121–126] along with binding sites

for FAD and NADPH [114]. Furthermore, Rho-GTPase regu-lation of Nox proteins is comparable with its regulation inphagocytes, i.e., gp91phox (Nox2) regulation [114]. Moreover,nonphagocytic cells contain homologs to p47phox and p67phox,known as p41 and p51, respectively, which can regulate Noxproteins, specifically Nox1 [127]. Specific regulation of O2

–

generation has been located in the charged amino acids in theD-loop region and in the �-helical loop of the C terminus [128].The regulation of the NADPH oxidase and the generation ofROS are most noted in PMNs and leukocytes; therefore, moreresearch needs to done to fully delineate the regulation ofNADPH oxidase activity and the correlation between phago-cytic and nonphagocytic cells.

CYTOSOLIC COMPONENTS OF THENADPH OXIDASE

p47phox

The 390 amino acid peptide p47phox [129] contains four knowndomains: 1) an N-terminus phox homology (PX) domain, 2)tandem internal SH3 domain, 3) an autoinhibitory domain, and4) a C-terminus proline-rich domain [56, 130–134]. The PXdomain targets p47phox to the plasma membrane, where itinteracts with phosphatidylinositol-3/3,4/3,4,5-phosphate[PI(3/3,4/3,4,5)P] and an adjacent phosphatidic acid (PA) orphosphatidylserine [135–137]. In resting PMNs, the tandemSH3 domain binds to the autoinhibitory domain, masking thePX and proline-rich domains, which enable binding to p67phox

and p40phox [138, 139]. Phosphorylation of p47phox is not onlya prerequisite for translocation to the membrane, but in mostcases, it also permits direct interaction with p22phox, therebyfacilitating the binding of p40phox and p67phox to cytochromeb558 [140, 141]. The C-terminus proline-rich region contains atleast six potential serine phosphorylation targets for PKC andtwo mitogen-activated protein kinase (MAPK) phosphorylationsites [142–144]. With regards to PKC phosphorylation, theisoforms �, �, �, and � have been shown to cause the phos-phorylation and translocation of p47phox with nonphysiologicstimuli, such as phorbol myristate acetate (PMA) in primaryand transformed cell lines [145–147].

One form of CGD is a deficiency in p47phox from mutationson the p47phox protein, which include a GT or TG deletion atexon 2 [148–150] or an Arg-to-Gln mutation in the PX do-mains of [135]. In p47phox-deficient CGD, p67phox, p40phox, andRac2 are unable to translocate from the cytosol to the mem-brane with formyl-methionyl-leucyl-phenylalanine (fMLP) orPMA activation [53, 151]. The characteristics of p47phox-defi-cient CGD are mimicked in p47phox�/� mice [152, 153].However, unlike in CGD, the activation of the NADPH oxidasein some cell-free systems does not always require the translo-cation of p47phox [154–156], but it is needed for maximaloxidase activity.

p67phox

The 526 amino acid peptide [129] p67phox contains five knowndomains: 1) four N terminus tetratricopeptide repeat domains(TPRs), which interact with Rac [133]; 2) an activation domain,

Sheppard et al. Structural organization of the PMN oxidase 1027

which binds to gp91phox following phosphorylation and trans-location [127]; 3) an internal praline-rich domain [133]; 4) twoSH3 domains on the N-terminus side, which can bind to theproline-rich domain of p47phox in resting and activated PMNs[53, 129, 157, 158]; and 5) the PB1 domain, a 150 amino acidstretch between the two SH3 domains and the site of theC-terminus PC motif for p40phox interaction [159–161]. Thep67phox-p40phox interaction is maintained even in the presenceof anionic amphiphiles, raising the possibility that this inter-action mediates a constitutive association in resting and acti-vated cells [160]. Phosphorylation of p67phox is not only aprerequisite for translocation from the cytosol to the membranebut is also required for the translocation of p40phox and Rac2 tothe membrane [151, 162]. Also, p67phox contains a catalyticNADPH binding site for electron transfer to the FAD in thecytochrome b558 complex [50, 155, 163, 164]. More specifi-cally, amino acids 1–210 of p67phox are required for a steady-state reduction of FAD, indicating a dominant effect on hy-dride/electron transfer, and amino acids 199–210 are impor-tant for regulating the electron flow [50]. The dehydrogenation(reduction) activity of NADPH by p67phox is proportional to theenzyme concentration, independent of FAD, insensitive to O2

–

dismutase, and inhibited by high concentrations of ferricyanide[165]; moreover, these findings suggest that p67phox is involvedin the transfer of electrons between NADPH and the oxidaseflavin [165].

CGD with a deficiency in p67phox is rare and results in adecrease in oxidase production and immunosuppression [166,167]. Various mutations have been identified that result in theabsence of p67phox [168], such as a mutation that results in adeletion of the AAG sequence along with a deletion in anotherallele [169] or a T-to-C point mutation in intron 3 of the p67phox

prevents the accumulation of p67phox mRNA [170]. A defi-ciency of p67phox in CGD fails to translocate p40phox in re-sponse to PMA, also confirmed by cell-free assays [154, 171],whereas p47phox translocation and phosphorylation are unaf-fected [172–174].

p40phox

The p40phox protein is 339 amino acids in length [175], itsexpression is restricted to hematopoietic cells, with the excep-tion of erythroid cells [176], and was originally identified as aprotein tightly associated with p67phox [177, 178]. There arethree known domains on p40phox: 1) an N terminus PX domain,specific for PI(3)P, 2) an internal SH3 domain, and 3) aC-terminus PC motif, which can interact with the PB1 domainof p67phox [133, 137, 161, 175, 178, 179]. In resting cells,p40phox is basally phosphorylated, but the onset and extent ofadditional phosphorylation strongly correlate with the level ofO2– generation [180]. Phosphorylation of p40phox occurs on theSer315 and Thr154 residues and is inhibited markedly by thePKC inhibitor H-7, indicating PKC as a possible direct phos-phorylator of p40phox in the p40phox-p47phox-p67phox trimer[173, 181]. Additionally, p40phox appears to down-regulateoxidase function by competing with the SH3 domain interac-tions between other essential oxidase components [172].

There have not been any p40phox defects or deficienciesdemonstrated to date; however, in CGD patients lackingp67phox, the amount of cytosolic p40phox is decreased signifi-

cantly [151, 174, 175]. Therefore, the exact functional role ofp40phox in oxidase activation and regulation has not been fullyelucidated and may be dependent on multiple variables.

Rac 1/2

Rac is a small G-protein (21 kDa) member of the Ras super-family, which is required for activation of the NADPH oxidase[37, 109, 182, 183]. In resting neutrophils, Rac2, the primaryisoform in human PMNs [98%; although Rac1 is also present(2%)], is located in the cytosol [95, 151, 183]. Upon activation,Rac2 rapidly converts from a GDP- to GTP-bound state, dis-sociates from Rho guanine-nucleotide-dissociation inhibitor,and migrates to the membrane [184, 185]. Rac2 (amino acidresidues 170–199) binds directly to p67phox at the membranein a 1:1 stoichiometry and a dissociation constant value of 60nM, but it does not bind to p40phox or p47phox [109, 186, 187].The N-terminus (amino acids 1–192) of p67phox can be used asa specific inhibitor of Rac2 signaling, reducing the ability ofRac2-GTP to disrupt the p67phox-p40phox binding [188, 189].Moreover, mutational studies have identified two regions inRac2, which are important for activity: 1) the “effector region”(residues 26–45) and 2) the “insert region” (residues 124–135) [164, 190]. Proteins mutated in the effector region (N26H,I33N, and D38N) inhibit Rac2 binding to p67phox, and theinsert region mutations (K132E and L134R) bind with normalaffinity to p67phox [182]. The motif RQQKRP in the C-terminusregion and the D150 amino acid have recently been identifiedas being essential for O2

– production, chemotaxis, and F-actinassembly [191]. Based on this information, a model is postu-lated, whereby the Rac2 effector region binds p67phox, theRac2 C-terminus binds to the membrane, and the insert regionlikely interacts with cytochrome b558.

Rac2 deficiency has been implicated in CGD. Rac2 defi-ciency has been mainly studied in mice and has been shown tobe important for degranulation, chemotaxis, actin formation,and O2

– production [192–195]. PMNs from Rac2�/� mice havedecreased responsiveness to activating stimuli [i.e., fMLP,leukotriene B4 (LTB4), complement 5a (C5a)], although p67phox

still binds to the membrane, but still do not produce O2– or

release MPO or elastase [196–199]. In humans, however, aD57N point mutation was identified in a patient, which re-sulted in the inability of GTP to bind to Rac2, although GDPbinding was normal, and decreased O2

– production was ob-served [200]. In cell-free systems, the importance of Rac2 inthe activation of the NADPH oxidase system has been shown[107, 109, 201].

Cdc42

Cdc42 is a small G-protein with 70% homology to Rac1/2[202]. The main difference is in the effector domain, alsoknown as Switch I, where there is a four amino acid difference[201–203]. The two amino acids that have the most effect,however, are amino acids 27 and 30, which when mutated inthe Cdc42 protein, become fully functional in activating theoxidase, similar to Rac2 [202–204]. Thus, Cdc42, which canbe stimulated by �2-integrins [205], acts as an inhibitor ofoxidase activation. Recently, it was suggested that Rac andCdc42 act as antagonists, competing through the insert domain


for binding to cytochrome b558 or, more specifically, the trans-located p67phox [201].

p29 peroxiredoxin

This is a newly discovered oxidase-associated protein, whichcoimmunoprecipitated with p67phox [39]. It has phospholipaseand peroxidase activity and when preincubated with recombi-nant cytosolic oxidase proteins Rac1, p47phox, and p67phox,increased oxidase activity [39]. It is thought that p29 perox-iredoxin has an effect on the oxidase by its peroxiredoxinactivity as a result of its ability to reduce thioredoxin [39].Further studies must be conducted to elucidate the role of thep29 peroxiredoxin as an oxidase-associated protein; there arecurrently no known deficiencies of this protein.

THE PMN STAGES FOR NADPH OXIDASEACTIVITY: RESTING, PRIMED,AND ACTIVATED

Human PMNs function in host defense against microbial in-vaders by migration to various tissues. PMNs exhibit threedifferent phenotypes, which are dependent on external stimuli.The first phenotype may be classified as resting or quiescent,when PMNs are freely flowing in the circulation and have around morphology with minimal membrane ruffling [206, 207].In the vascular endothelium, a proinflammatory stimulus in-duces the PMNs to change from the nonadherent, quiescentphenotype to an adherent phenotype, secondary to chemokinerelease on the surface of the endothelium [206, 207]. ThePMNs first roll through selectin-mediated interactions, thenrapidly adhere to the endothelium, and are primed at thisjuncture [206, 207]. Priming not only changes the PMN shapeand allows for adherence, but PMNs become functionally hy-per-reactive in that stimuli, which normally would not cause

activation and release of the microbicidal arsenal but nowcause degranulation and oxidase activity of the primed, firmlyadherent PMNs [65, 206, 207]. These findings have formed thebasis for the pathophysiology of ALI and other cytotoxic effectscaused by PMNs [7, 65, 208–210]. Moreover, the adherentPMN then diapedeses through the endothelial layer and che-motaxis along a gradient of chemoattractant mediators until itreaches the nidus of infection in the tissue [206, 207]. At thesite of infection, PMNs phagocytose bacteria with subsequentassembly and activation of the oxidase at the phagolysosome[206, 207, 211].

The complexities of NADPH oxidase are many-fold, andfurther elucidation of the various signaling circuitry, kinases,phosphatases, and lipases involved will be required before firmstatements regarding the precise mechanism(s) underlying invivo oxidase priming/activation can be made. However, each ofthese phenotypes (resting, primed, and activated) will be dis-cussed below as they relate to the cellular structure (cytosol,membrane, and granule) important for the NADPH oxidase.

RESTING PMNs (Fig. 1)

Cytosol

Initial studies found that in resting neutrophils, the cytosolicsubunits p40phox-p67phox-p47phox exist in a heterotrimeric com-plex with a 1:1:1 ratio [156, 179, 212, 213]; thus, the highmolecular weight of this complex is a result of an extended,nonglobular shape rather than the presence of multiple copiesof any of the proteins [212]. However, more recent studies havedemonstrated that there exists stoichiometrically distinct poolsof the oxidase components: 1) dimerization between p67phox

and p47phox (p67phox-p47phox), 2) dimerization between p40phox

and p67phox (p40phox-p67phox), 3) trimer formation between

Fig. 1. Resting membrane of human PMNs. Rep-resentative picture of a resting PMN and the loca-tion of the NADPH oxidase components, includingthe cytosolic components (p47phox, p67phox, p40phox,Rac2, and p29) and the membrane-bound compo-nents (gp91phox, p22phox, and Rap1A). In the restingPMN, the cytosolic units, excluding Rac2, are com-plexed together, and Rac2 is inactive with thebound GDP. The membrane units, which form cy-tochrome b558, are found in the plasma membraneand in the membrane of the specific granules andsecretory vesicles. RHOGDI, Rho-GDP dissocia-tion inhibitors.


p40phox and p67phox and p47phox (p47phox-p67phox-p40phox),where p67phox acts as a “bridge” between p40phox and p47phox,and 4) as monomers, as seen with p47phox [140, 212, 214, 215].

Membrane

The membrane in resting PMNs contains a small portion ofgp91phox and p22phox, along with the FAD and heme moietiesof the cytochrome b558 [53, 213, 216, 217]. Also, in somestudies using Trition X-100 for subcellular fractions, p67phox,p40phox, gp91phox, and p22phox were found in the insolublefraction, or the cytoskeleton [218, 219]. However, in nondeter-gent, subcellular fractions, the p67phox and p40phox were lo-cated in the cytosol, as described above.

Granules

The specific granules and the secretory vesicles contain themajority of the gp91phox and p22phox [53, 213, 216, 220].

THE PRIMED PMN (Fig. 2)

Priming of the NADPH oxidase is defined operationally asaugmentation of O2

– generation in response to a second, acti-vating stimulus [221, 222]. Various chemoattractants serve aspriming agents by changing the PMN phenotype from nonad-

herent to adherent; however, operationally, priming does notcause the activation of the NADPH oxidase. There are twoclassifications of priming agents: rapid [PAF, LTB4, C5a, ly-sophosphatidylcholine (LPC)], which acts in 3–5 min andusually involves a tyrosine kinase, and long-acting [LPS, tumornecrosis factor-� (TNF-�), GM-CSF, IL-18], which takes15–60 min to manifest their effect [141, 211, 223–227]. Dif-ferent priming agents have a range of effects on the structuralorganization of the oxidase and will be reviewed briefly below.

Cytosol and membrane

Binding of a “priming” agent (e.g., PAF, LPC, C5a, IL-8, orLPS) to a G-protein-coupled receptor (GPCR) on PMNs may,pertaining to the oxidase, take from seconds to several minutesto up to 2 h (e.g., interferon-) for maximal augmentation to asubsequent stimulus and thus, priming of the PMN [209, 211,225, 226, 228–231]. Priming of the oxidase with long-actingpriming agents, such as LPS, GM-CSF, and IL-18, translocatesp47phox to the plasma membrane [141, 211, 231]. It is thoughtthat the degree of p47phox phosphorylation, a prerequisite fortranslocation, correlates with the rigor of the priming agent[232, 233]; however, with TNF-� priming (15–60 min), there isonly partial phosphorylation of p47phox, not translocation, butTNF-� is still considered an effective priming agent [224, 231,233–235]. Conversely, PAF, a rapid (3–5 min) primer, phos-

Fig. 2. Primed membrane of human PMNs. Representative image of the phosphorylation and translocation of the oxidase components with external priming agents,such as interleukin (IL)-18, granulocyte macrophage-colony stimulating factor (GM-CSF), and lipopolysaccharide (LPS), only translocate p47phox to the plasma orgranular membrane (A), and other stimuli, such as homocysteine, angiotensin II, and C5a, translocate p47phox-p67phox to the plasma (granular) membrane (B).Another possibility is that p67phox, but not p47phox, translocates to the membrane with a stimulus, e.g., platelet-activating factor (PAF; C). ATP, Adenosine5�-triphosphate; ADP, adenosine 5�-diphosphate; GEF, guanine nucleotide exchange factors.


phorylates p67phox, p40phox, and Rac2, but not p47phox [234].Preliminary data suggest that p67phox then translocates to theplasma membrane with PAF priming [236]. When PMNs areincubated with LPS (30–60 min), there is an increase in theassociation of cytochrome b558 with the plasma membrane alongwith phosphorylation and translocation of p47phox, but not p67phox,p40phox, or Rac2 [141]. However, other priming agents, such ashomocysteine, angiotensin II, opsonized zymosan (OpZ), and stim-ulation through the �2-integrins, will cause phosphorylation ofp47phox and p67phox [219, 237–239]. Phosphorylation and trans-location of the cytosolic oxidase components to the plasma mem-brane during priming augment oxidase activity when a secondstimulus is used [141, 211, 222, 225, 227, 231, 240–242].

Along with the influence of the cytosolic oxidase componentson priming, this phenomenon is regulated by additional pro-teins, which are vital to the various signaling pathways. One ofthe key regulators is from the family of MAPKs, specifically,p38 MAPK and extracellular signal-regulated kinase (Erk)1/2(p42 and p44 MAPK, respectively). Many studies have shownthat when either protein is inhibited, O2

– production is inhib-ited [144, 234, 237, 238, 243–249]. The exact mechanism forp38 MAPK and Erk1/2 phosphorylation of the cytosolic oxi-dase components, especially p40phox, p67phox, and p47phox, hasyet to be understood fully, but there may be a dual role in PMNoxidase release. TNF-� priming of human and porcine PMNsactivates p38 MAPK and directly phosphorylates p47phox andp67phox [234, 243, 250, 251], whereas with PAF priming, thereis only p67phox phosphorylation through p38 MAPK activation[234]. Other priming agents, such as ionomycin and angioten-sin II, activate p38 MAPK and Erk1/2 [238, 252], However,p38 MAPK and Erk1/2 do not always phosphorylate the oxi-dase components directly, but instead, these MAPKs can ac-tivate a secondary protein or lipid mediator in a signalingpathway [144, 253–257].

Lipid mediators have been implicated in priming of theNADPH oxidase. LPC, arachidonic acid (AA), and the leukotri-enes, specifically LTB4, have been shown to cause an increase inO2

– release in response to a subsequent stimulus [209, 223, 228,241, 253, 254, 256, 258–263]. The exact mechanism of action isstill relatively unknown, but it appears that there is involvement ofa G-protein [probably G�i, based on pertussis toxin (PTX) inhibi-tion] and the MAPK pathway [221, 241, 257, 264–267]. LTB4 hasbeen shown to activate Erk1/2 [210, 268, 269] and cause thetranslocation of Rac2 [270]. Also, these lipid mediators mayactivate PKC and phosphoinositide-3 kinase (PI-3K) and areCa2�-dependent [110, 142–144, 215, 225, 254, 271, 272]. Fur-ther studies are required to determine the exact mechanism bywhich these lipids signal.

Another key component in human PMN priming is cytosoliccalcium. The activation of many of the proteins required forpriming is calcium-dependent, such as PKC, MAPK, and ty-rosine kinases [211, 238, 241, 245, 247, 273–279]. The influxof calcium to the cytosol from the internal and external storescreates a voltage gradient and a change in the membranepotential [280]. The rise in cytosolic calcium triggers the startof many signaling cascades essential for priming and assemblyof the oxidase components [222, 241, 247, 252, 281, 282].Inhibiting calcium release or influx not only inhibits the prim-ing of PMNs but also activation of the oxidase [241, 252, 282].

Priming of PMNs changes the physical structure of the cellby the rearrangement of actin, especially in F-actin. The actincytoskeleton may provide a means for coordinating the processof NADPH oxidase assembly [80, 283–286]. Currently, exactmechanisms of oxidase activation, with respect to cytoskeletonreorganization, are not fully understood. Initial studies indicatean association with the cytoskeleton based on the phox proteindetergent insolubility in whole cells or at the membrane [69,142]. In human PMNs, the phox proteins particularly seem tointeract with coronin, a cytoplasmic, actin-associated proteininvolved in the dynamics of the actin system, at the cytoskel-eton [287–289]. Coronin is selectively solubilized when thePMN oxidase is activated by fMLP or PMA; however, it is notsolublized in the absence of cytochrome b558 [290]. In addition,p67phox copurifies with coronin via the C-terminus half ofp40phox and accumulates around the phagocytic cup in PMNs[218, 290–292]. Moesin, an F-actin-binding protein, binds top47phox and p40phox in a phosphoinositide-dependent mannervia the N-terminal of the PX domains [218, 293]. In addition,cofilin has been found to associate with p67phox [294]. Cofilindephosphorylation occurs following PMA and fMLP stimula-tion of PMNs, and the kinetics are similar to those observed forO2

– generation. Coflin dephosphorylation is also associated withmovement toward F-actin-rich areas at the cell periphery [218,295, 296] and colocalizes with areas high in ROS generation,implicating a role for cofilin and F-actin in oxidase activity [218,296]. Oxidase subunit association with actin or actin-bindingproteins has also been confirmed by microscopy and Western blotanalysis [218, 288, 295, 297–300]. However, more studies, espe-cially with digital microscopy of whole, fixed PMNs, need to becompleted to better understand the interaction amongst actin, theoxidase components, and membrane rearrangement.

There is increasing evidence that signal transduction inPMNs may occur in multiprotein signalosomes, or preas-sembled signaling complexes [301, 302] and may regulateactions on a spatio-temporal basis. An obvious requisite is theassembly of some kind of backbone, a classic being the as-sembly of the cytoskeleton. However, unlike the cytoskeleton,which may support assembly on a more global level, clatherin,caveolin, and the emergence of lipid domains serve as specificmicrodomains, assembling at and as in a response to externalstimuli [303–305]. There are two major concerns for PMNsignaling, oxidase assembly, and the subsequent release ofO2

–. First, there is evidence for assembly in the region ofreceptor ligation, which is internalized via clathrin-mediatedendocytosis [306–308] or lipid rafts {Fc receptors (FcRs)[309–312]}. Second, human PMNs do not contain caveolin[313], a significant subunit of lipid rafts, whereas many“model” cell lines do. The use of HL-60 cells, when properlydifferentiated, exhibits 90% homology to human PMNs, con-ferring their practical purpose [314]. However, when usingphagocytic cells of a different delineation (i.e., monocyte pre-cursors, U798, RAW39393, adipocytes, and endothelial deriv-atives) or even across different species of PMNs, this homologyis not the case. For example, when comparing human PMNs withbovine PMNs, one of the big differences is the presence ofcaveolin in bovine PMNs [315]. Therefore, it is important toconsider the cell linage when comparing in vitro and in vivostudies.


Granules

When a priming agent, such as LPS, binds to the PMN, theinternal granules (specific and azuraphilic granules and secre-tory vesicles) fuse with the plasma membrane to form a phago-some, thus allowing gp91phox and p22phox to interact with themembrane [73, 220, 316]. The increase of cytochrome b558 atthe plasma membrane as a result of degranulation is welldocumented [241, 317–320]; however, the role of the specificgranules in oxidase generation is still debatable. Upon incu-bation with PMA, the cytosolic oxidase components, p47phox,p67phox, and Rac2, translocate, not only to the plasma mem-brane but also the specific granules, where they are able toproduce O2

– for a short period of time [39, 74, 321]. Oxidaseactivity in the granules is dependent on PKC� and PI-3Kactivity to allow for proper assembly of the oxidase components[215, 322]. Also, the release of MPO and elastase from theazuraphilc granules during degranulation is important for thephagocytic activity of PMNs [323–327].

THE ACTIVATED PMN (Fig. 3)

The activated phenotype of PMNs is a result of activation of theNADPH oxidase with complete assembly of all the oxidase

components at the membrane (the plasma or granular) and theexchange of electrons across the membrane, culminating in therelease of O2

–. Common agents used for activation are fMLP,PMA, and OpZ, each with different signaling pathways but allresulting in the activation of the NADPH oxidase. It should benoted that some of the agents used for priming PMNs can alsocause activation if used in a high enough, although not alwaysphysiological, concentration [65, 247, 328]

Cytosol

Activation of oxidase requires additional phosphorylation,mainly on the serine and threonine residues of p47phox,p67phox, and p40phox, via kinases such as PKC, p21-activatedkinase (PAC), p38 MAPK, PI-3K, and PA-activated proteinkinase [45, 78, 144, 329, 330], followed by translocation to themembrane. Activation of the NADPH oxidase also requires thedissociation of p40phox from p67phox via the association of anactivated Rac2 with p67phox [174, 189]. However, blocking thebond between p40phox and p67phox inhibits cell-free oxidaseactivation but not the translocation of the cytosolic proteins,including p47phox to the membrane, nor does it inhibit acti-vated oxidase activity in vivo [178]. In some cases, tyrosinephosphorylation is important for oxidase activation, such as theFcR for immunoglobulin G IIA (FcRIIA)-stimulated phosphor-

Fig. 3. Activated membrane of human PMNs. Representative image of the complete translocation of the oxidase components to the plasma or granular membraneand the electron transfer across the membrane, characteristic of activation of the NADPH oxidase. All of the oxidase components are complexed at the plasma(granular) membrane, which initiates the reduction of NADPH to NADP and the generation of two electrons, which flow across the membrane via FAD and thengenerate the O2

– in a phagolysosome. The O2– produces other bactericidal products, such as H2O2.


ylation of p72syk as an early signal that precedes activation[330, 331]. Activation of PMNs results in complete transloca-tion of all cytosolic oxidase components to the plasma orvesicle membrane.

Membrane

Activation of the oxidase through binding of soluble ligands(i.e., fMLP) to surface receptors usually results in O2

– produc-tion lasting less than 5 min, whereas receptor-independentactivation (i.e., PMA) causes prolonged generation of the O2

–

until depletion of necessary substrates and cofactors [332].Furthermore, in intact cells, the activated oxidase is experi-encing a continuous process of activation and deactivation[333].

Complete assembly of the oxidase components at the mem-brane is the final stage of activation. As previously establishedwith priming, gp91phox and p22phox are already at the mem-brane, and there is translocation and partial phosphorylation ofthe cytosolic components. It is well defined that the SH3domain of the p47phox interacts with the proline-rich domain ofp22phox [61, 77, 81, 134, 138, 334, 335]. The polyprolineregion of p47phox binds to the C-terminus SH3 domain ofp67phox [61, 81, 131, 138, 156], and the TPR region of p67phox

binds to Rac2 at its effector region; Rac2 binds to the mem-brane at its C-terminus, and the insert region probably inter-acts with gp91phox [140, 188, 336]. The SH3 domain of p40phox

binds to the polyproline region of p47phox, although p40phox

binds more tightly to p67phox, and p40phox acts as bridgebetween p47phox and p67phox at the membrane [156, 175, 177,179, 189, 212, 335]. Although Rap1A has been implicated inoxidase activity, its role in the oxidase complex is controversialbut may bind to the cytochrome b2 complex at its carboxyterminus [38, 105, 108, 213, 337]. The PX domain of p40phox

and p47phox also interacts with F-actin-binding proteins at themembrane via phosphoinositol-mediated signaling [177, 212,290, 293]. These interactions are proposed to be constant,regardless of the activating factor.

PMA has become the most commonly used positive controlfor oxidase activation [332, 338]; however, it is a nonphysi-ological stimulus. Originally considered to be involved in tu-mor promotion, although by themselves were not carcinogenic,phorbol esters resulted in many genetic and phenotypicchanges, including an increase in oxidase production and thecopurification of PKC with the putative phorboid receptor[339–343]. Thus, PMA is a PKC agonist. Moreover, it ispossible that the efficacy of ROS production in response toPMA has overridden its own irrelevancy in in vivo PMNfunction. The response to multiple stimuli (i.e., fMLP) over areasonable amount of time will exhibit multiple oxidative re-sponses; however, PMA uses all available resources in theproduction of ROS. Essentially, PMA, once the substrates arediminished, lacks any further oxidative capacity in the PMN,whereas other activators may allow for multiple oxidative re-sponses. This is important to consider, as the lifespan of PMNsis 24 h in vivo and 12 h in vitro.

The bacterial peptide fMLP is another oxidase activator thatsignals in a receptor-dependent mechanism. The fMLP recep-tor, a GPCR, regulates various activities in PMNs via a PTX-sensitive G-protein, which includes chemotaxis/chemokinesis,

exocytosis (degranulation) [330], and multiple signaling path-ways including MAPK, lipid kinases, the production of secondmessengers by various phospholipases (e.g., PLC, PLA2, andPLD) [4, 5], and the activation of PKC [11, 344–346]. Forexample, the binding of fMLP to its receptor activates PLC,which generates DAG and inositol trisphosphate and the re-lease of intracellular Ca2� [271, 347–349]. DAG can also beconverted to PA by the action of a stimulus-responsive, trans-locatable diacylglycerol kinase [350] or through the MAPKactivation of PLD [351–354], which is involved in oxidaseactivation [355, 356]. PMNs that are treated sequentially withLPS (priming) and fMLP (activation) have a three- to sixfoldincrease (compared with either agent alone) in the plasmamembrane content of p47phox, p67phox, and Rac2 and aug-mented O2

– generation by intact PMNs.OpZ has also been shown to activate the NADPH oxidase

and cause phagocytosis in PMNs; however, unlike PMA, whichactivates PKCs, OpZ activates cytosolic PLA2 (cPLA2) and therelease of AA for oxidase activation [338, 357, 358] and anefflux of protons [359, 360], with further regulation by Erk1/2.The absence of cPLA2 inhibits O2

– generation [261], but theaddition of AA restores O2

– generation and efflux of protons[261, 361]. Moreover, Erk1/2 and p38 MAPK are required forthe onset of cPLA2 activation through the engagement of thetyrosine kinase Pyk2 [362]; however, activation of the protonchannel and the NADPH oxidase is unaltered by the presenceof COX and lipoxygenase inhibitors, indicating that the open-ing of the proton channel and the activation of O2

– generationare not mediated via AA metabolites of these enzymes [362].OpZ has also been shown to activate the C3 receptor and theFcR [363, 364].

Granules

NADPH oxidase activation also occurs in the granules. Thecytosolic components translocate to the granular membranesfor the production of O2

–, which is probably for the intracel-lular destruction of bacteria. Oxidase generation could alsocontribute to the apoptotic signaling pathway [39, 73, 317].The activation of PMNs with fMLP and OpZ has a greaterresponse than with PMA and the formation of the NADPHcomplex, similar to the plasma membrane [73].

SUMMARY

Neutrophils are an essential component of the innate immunesystem via the respiratory burst involved in bactericidal activ-ity and the eradication of pathogens. In human PMNs, theNADPH oxidase exhibits three different phenotypes: resting,primed, and activated. Each of these phenotypes is importantfor the proper function of the oxidase to release the O2

– anion.There are physiological consequences of a malfunction in anyof these stages including CGD and activation of the oxidativemicobicidal arsenal in inappropriate locations, i.e., the micro-vasculature, resulting in injury to the lung and other organs [1,241, 328, 365, 366]. For example, PMNs from severely injuredpatients display an increased oxidative response along with anincreased ability to sequester and concentrate in various end


organs, which can result in multiple organ failure [365, 366].Furthermore, LPCs, effective primers of the PMN oxidase[241], will increase the bactericidal activity of PMNs in miceby increasing the production of H2O2 and increasing survivalin a sepsis model of cecal ligation and puncture [367]. Furtherinvestigation is needed to elucidate signaling mechanisms inthe function of the NADPH oxidase and its role in host defensein the congruence of component translocation and in opposingsignaling mechanisms. Moreover, studies need to focus onintact PMNs to preclude spurious protein–protein interactionsas a result of techniques that use detergents, especially withrespect to the cytoskeleton, or artificially primed PMNs duringisolation from whole blood.

REFERENCES

1. Babior, B. M. (2000) Phagocytes and oxidative stress. Am. J. Med. 109,33–44.

2. Baldridge, C. W., Gerard, R. W. (1933) The extra respiration of phago-cytosis. Am. J. Physiol. 103, 235–236.

3. Sbarra, A. J., Karnovsky, M. L. (1959) The biochemical basis of phago-cytosis. I. Metabolic changes during the ingestion of particles by poly-morphonuclear leukocytes. J. Biol. Chem. 234, 1355–1362.

4. Selvaraj, R. J., Sbarra, A. J. (1966) Relationship of glycolytic andoxidative metabolism to particle entry and destruction in phagocytosingcells. Nature 211, 1272–1276.

5. Holmes, B., Page, A. R., Good, R. A. (1967) Studies of the metabolicactivity of leukocytes from patients with a genetic abnormality of phago-cytic function. J. Clin. Invest. 46, 1422–1432.

6. Wittels, E. H., Coalson, J. J., Welch, M. H., Guenter, C. A. (1974)Pulmonary intravascular leukocyte sequestration. A potential mechanismof lung injury. Am. Rev. Respir. Dis. 109, 502–509.

7. Ratliff, N. B., Wilson, J. W., Mikat, E., Hackel, D. B., Graham, T. C.(1971) The lung in hemorrhagic shock. IV. The role of neutrophilicpolymorphonuclear leukocytes. Am. J. Pathol. 65, 325–334.

8. Clark, R. A. (1990) The human neutrophil respiratory burst oxidase.J. Infect. Dis. 161, 1140–1147.

9. Allen, R. C., Yevich, S. J., Orth, R. W., Steele, R. H. (1974) Thesuperoxide anion and singlet molecular oxygen: their role in the micro-bicidal activity of the polymorphonuclear leukocyte. Biochem. Biophys.Res. Commun. 60, 909–917.

10. Babior, B. M., Peters, W. A. (1981) The O2-producing enzyme of humanneutrophils. Further properties. J. Biol. Chem. 256, 2321–2323.

11. DeLeo, F. R., Quinn, M. T. (1996) Assembly of the phagocyte NADPHoxidase: molecular interaction of oxidase proteins. J. Leukoc. Biol. 60,677–691.

12. Roberts, J., Camacho, Z. (1967) Oxidation of NADPH by polymorpho-nuclear leucocytes during phagocytosis. Nature 216, 606–607.

13. Babior, B. M. (1982) The enzymatic basis for O-.2 production by humanneutrophils. Can. J. Physiol. Pharmacol. 60, 1353–1358.

14. Hampton, M. B., Kettle, A. J., Winterbourn, C. C. (1998) Inside theneutrophil phagosome: oxidants, myeloperoxidase, and bacterial killing.Blood 92, 3007–3017.

15. Dallegri, F., Ballestrero, A., Frumento, G., Patrone, F. (1986) Role ofhypochlorous acid and chloramines in the extracellular cytolysis byneutrophil polymorphonuclear leukocytes. J. Clin. Lab. Immunol. 20,37–41.

16. Shepherd, V. L. (1986) The role of the respiratory burst of phagocytes inhost defense. Semin. Respir. Infect. 1, 99–106.

17. Thomas, E. L., Lehrer, R. I., Rest, R. F. (1988) Human neutrophilantimicrobial activity. Rev. Infect. Dis. 10 (Suppl. 2), S450–S456.

18. Babior, B. M., Kipnes, R. S., Curnutte, J. T. (1973) Biological defensemechanisms. The production by leukocytes of superoxide, a potentialbactericidal agent. J. Clin. Invest. 52, 741–744.

19. Dunford, H. B. (1987) Free radicals in iron-containing systems. FreeRadic. Biol. Med. 3, 405–421.

20. Heinecke, J. W., Li, W., Daehnke III, H. L., Goldstein, J. A. (1993)Dityrosine, a specific marker of oxidation, is synthesized by the my-eloperoxidase-hydrogen peroxide system of human neutrophils and mac-rophages. J. Biol. Chem. 268, 4069–4077.

21. Marquez, L. A., Dunford, H. B. (1995) Kinetics of oxidation of tyrosineand dityrosine by myeloperoxidase compounds I and II. Implications forlipoprotein peroxidation studies. J. Biol. Chem. 270, 30434–30440.

22. Klebanoff, S. J. (1968) Myeloperoxidase-halide-hydrogen peroxide anti-bacterial system. J. Bacteriol. 95, 2131–2138.

23. Davis, W. B., Mohammed, B. S., Mays, D. C., She, Z. W., Mohammed,J. R., Husney, R. M., Sagone, A. L. (1989) Hydroxylation of salicylate byactivated neutrophils. Biochem. Pharmacol. 38, 4013–4019.

24. Tauber, A. I., Babior, B. M. (1977) Evidence for hydroxyl radicalproduction by human neutrophils. J. Clin. Invest. 60, 374–379.

25. Weiss, S. J., Rustagi, P. K., LoBuglio, A. F. (1978) Human granulocytegeneration of hydroxyl radical. J. Exp. Med. 147, 316–323.

26. Eiserich, J. P., Hristova, M., Cross, C. E., Jones, A. D., Freeman, B. A.,Halliwell, B., van der Vliet, A. (1998) Formation of nitric oxide-derivedinflammatory oxidants by myeloperoxidase in neutrophils. Nature 391,393–397.

27. Dinauer, M. C., Pierce, E. A., Bruns, G. A., Curnutte, J. T., Orkin, S. H.(1990) Human neutrophil cytochrome b light chain (p22-phox). Genestructure, chromosomal location, and mutations in cytochrome-negativeautosomal recessive chronic granulomatous disease. J. Clin. Invest. 86,1729–1737.

28. Kunkel, L. M., Monaco, A. P., Middlesworth, W., Ochs, H. D., Latt, S. A.(1985) Specific cloning of DNA fragments absent from the DNA of a malepatient with an X chromosome deletion. Proc. Natl. Acad. Sci. USA 82,4778–4782.

29. Volpp, B. D., Nauseef, W. M., Donelson, J. E., Moser, D. R., Clark, R. A.(1989) Cloning of the cDNA and functional expression of the 47-kilo-dalton cytosolic component of human neutrophil respiratory burst oxi-dase. Proc. Natl. Acad. Sci. USA 86, 7195–7199.

30. Nunoi, H., Rotrosen, D., Gallin, J. I., Malech, H. L. (1988) Two forms ofautosomal chronic granulomatous disease lack distinct neutrophil cytosolfactors. Science 242, 1298–1301.

31. Volpp, B. D., Nauseef, W. M., Clark, R. A. (1988) Two cytosolicneutrophil oxidase components absent in autosomal chronic granuloma-tous disease. Science 242, 1295–1297.

32. Lomax, K. J., Leto, T. L., Nunoi, H., Gallin, J. I., Malech, H. L. (1989)Recombinant 47-kilodalton cytosol factor restores NADPH oxidase inchronic granulomatous disease. Science 245, 409–412.

33. Leto, T. L., Lomax, K. J., Volpp, B. D., Nunoi, H., Sechler, J. M.,Nauseef, W. M., Clark, R. A., Gallin, J. I., Malech, H. L. (1990) Cloningof a 67-kD neutrophil oxidase factor with similarity to a noncatalyticregion of p60c-src. Science 248, 727–730.

34. Ambruso, D. R., Bolscher, B. G., Stokman, P. M., Verhoeven, A. J., Roos,D. (1990) Assembly and activation of the NADPH:O2 oxidoreductase inhuman neutrophils after stimulation with phorbol myristate acetate.J. Biol. Chem. 265, 924–930.

35. Clark, R. A., Volpp, B. D., Leidal, K. G., Nauseef, W. M. (1990) Twocytosolic components of the human neutrophil respiratory burst oxidasetranslocate to the plasma membrane during cell activation. J. Clin. Invest.85, 714–721.

36. Gabig, T. G., Crean, C. D., Mantel, P. L., Rosli, R. (1995) Function ofwild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60cell NADPH oxidase activation. Blood 85, 804–811.

37. Quinn, M. T., Parkos, C. A., Walker, L., Orkin, S. H., Dinauer, M. C.,Jesaitis, A. J. (1989) Association of a Ras-related protein with cyto-chrome b of human neutrophils. Nature 342, 198–200.

38. Quinn, M. T., Mullen, M. L., Jesaitis, A. J., Linner, J. G. (1992)Subcellular distribution of the Rap1A protein in human neutrophils:colocalization and cotranslocation with cytochrome b559. Blood 79,1563–1573.

39. Ambruso, D. R., Cusack, N., Thurman, G. (2004) NADPH oxidaseactivity of neutrophil-specific granules: requirements for cytosolic com-ponents and evidence of assembly during cell activation. Mol. Genet.Metab. 81, 313–321.

40. Gabig, T. G., Schervish, E. W., Santinga, J. T. (1982) Functional rela-tionship of the cytochrome b to the superoxide-generating oxidase ofhuman neutrophils. J. Biol. Chem. 257, 4114–4119.

41. Segal, A. W., Jones, O. T. (1979) The subcellular distribution and someproperties of the cytochrome b component of the microbicidal oxidasesystem of human neutrophils. Biochem. J. 182, 181–188.

42. Quinn, M. T., Mullen, M. L., Jesaitis, A. J. (1992) Human neutrophilcytochrome b contains multiple hemes. Evidence for heme associatedwith both subunits. J. Biol. Chem. 267, 7303–7309.

43. Segal, A. W., West, I., Wientjes, F., Nugent, J. H., Chavan, A. J., Haley,B., Garcia, R. C., Rosen, H., Scrace, G. (1992) Cytochrome b-245 is aflavocytochrome containing FAD and the NADPH-binding site of themicrobicidal oxidase of phagocytes. Biochem. J. 284, 781–788.


44. Doussiere, J., Brandolin, G., Derrien, V., Vignais, P. V. (1993) Criticalassessment of the presence of an NADPH binding site on neutrophilcytochrome b558 by photoaffinity and immunochemical labeling. Bio-chemistry 32, 8880–8887.

45. Babior, B. M. (1999) NADPH oxidase: an update. Blood 93, 1464–1476.

46. Rotrosen, D., Yeung, C. L., Leto, T. L., Malech, H. L., Kwong, C. H.(1992) Cytochrome b558: the flavin-binding component of the phagocyteNADPH oxidase. Science 256, 1459–1462.

47. Abo, A., Boyhan, A., West, I., Thrasher, A. J., Segal, A. W. (1992)Reconstitution of neutrophil NADPH oxidase activity in the cell-freesystem by four components: p67-phox, p47-phox, p21rac1, and cyto-chrome b-245. J. Biol. Chem. 267, 16767–16770.

48. Cross, A. R., Rae, J., Curnutte, J. T. (1995) Cytochrome b-245 of theneutrophil superoxide-generating system contains two nonidenticalhemes. Potentiometric studies of a mutant form of gp91phox. J. Biol.Chem. 270, 17075–17077.

49. Willis, D., Moore, A. R., Frederick, R., Willoughby, D. A. (1996) Hemeoxygenase: a novel target for the modulation of the inflammatory re-sponse. Nat. Med. 2, 87–90.

50. Cross, A. R., Curnutte, J. T. (1995) The cytosolic activating factorsp47phox and p67phox have distinct roles in the regulation of electronflow in NADPH oxidase. J. Biol. Chem. 270, 6543–6548.

51. Cross, A. R., Erickson, R. W., Curnutte, J. T. (1999) Simultaneouspresence of p47(phox) and flavocytochrome b-245 are required for theactivation of NADPH oxidase by anionic amphiphiles. Evidence for anintermediate state of oxidase activation. J. Biol. Chem. 274, 15519–15525.

52. DeLeo, F. R., Burritt, J. B., Yu, L., Jesaitis, A. J., Dinauer, M. C.,Nauseef, W. M. (2000) Processing and maturation of flavocytochromeb558 include incorporation of heme as a prerequisite for heterodimerassembly. J. Biol. Chem. 275, 13986–13993.

53. Heyworth, P. G., Curnutte, J. T., Nauseef, W. M., Volpp, B. D., Pearson,D. W., Rosen, H., Clark, R. A. (1991) Neutrophil nicotinamide adeninedinucleotide phosphate oxidase assembly. Translocation of p47-phox andp67-phox requires interaction between p47-phox and cytochrome b558.J. Clin. Invest. 87, 352–356.

54. Foroozan, R., Ruedi, J. M., Babior, B. M. (1992) The reduction ofcytochrome b558 and the activity of the respiratory burst oxidase fromhuman neutrophils. J. Biol. Chem. 267, 24400–24407.

55. Cross, A. R., Higson, F. K., Jones, O. T., Harper, A. M., Segal, A. W.(1982) The enzymic reduction and kinetics of oxidation of cytochromeb-245 of neutrophils. Biochem. J. 204, 479–485.

56. Babior, B. M. (1992) The respiratory burst oxidase. Adv. Enzymol. Relat.Areas Mol. Biol. 65, 49–95.

57. Ueno, I., Fujii, S., Ohya-Nishiguchi, H., Iizuka, T., Kanegasaki, S. (1991)Characterization of neutrophil b-type cytochrome in situ by electronparamagnetic resonance spectroscopy. FEBS Lett. 281, 130–132.

58. Fujii, H., Kakinuma, K. (1990) Direct measurement of superoxide anionproduced in biological systems by ESR spectrometry: a pH-jump method.J. Biochem. (Tokyo) 108, 983–987.

59. Fujii, H., Kakinuma, K. (1990) Studies on the superoxide releasing sitein plasma membranes of neutrophils with ESR spin-labels. J. Biochem.(Tokyo) 108, 292–296.

60. Foubert, T. R., Bleazard, J. B., Burritt, J. B., Gripentrog, J. M., Baniulis,D., Taylor, R. M., Jesaitis, A. J. (2001) Identification of a spectrallystable proteolytic fragment of human neutrophil flavocytochrome b com-posed of the NH2-terminal regions of gp91(phox) and p22(phox). J. Biol.Chem. 276, 38852–38861.

61. Vignais, P. V. (2002) The superoxide-generating NADPH oxidase: struc-tural aspects and activation mechanism. Cell. Mol. Life Sci. 59, 1428–1459.

62. Cross, A. R., Parkinson, J. F., Jones, O. T. (1984) The superoxide-generating oxidase of leucocytes. NADPH-dependent reduction of flavinand cytochrome b in solubilized preparations. Biochem. J. 223, 337–344.

63. Cross, A. R., Parkinson, J. F., Jones, O. T. (1985) Mechanism of thesuperoxide-producing oxidase of neutrophils. O2 is necessary for the fastreduction of cytochrome b-245 by NADPH. Biochem. J. 226, 881–884.

64. Light, D. R., Walsh, C., O’Callaghan, A. M., Goetzl, E. J., Tauber, A. I.(1981) Characteristics of the cofactor requirements for the superoxide-generating NADPH oxidase of human polymorphonuclear leukocytes.Biochemistry 20, 1468–1476.

65. Wyman, T. H., Bjornsen, A. J., Elzi, D. J., Smith, C. W., England, K. M.,Kelher, M., Silliman, C. C. (2002) A two-insult in vitro model of PMN-mediated pulmonary endothelial damage: requirements for adherenceand chemokine release. Am. J. Physiol. Cell Physiol. 283, C1592–C1603.

66. Ginsel, L. A., Onderwater, J. J., Fransen, J. A., Verhoeven, A. J., Roos,D. (1990) Localization of the low-Mr subunit of cytochrome b558 inhuman blood phagocytes by immunoelectron microscopy. Blood 76,2105–2116.

67. Cross, A. R., Jones, O. T., Harper, A. M., Segal, A. W. (1981) Oxidation-reduction properties of the cytochrome b found in the plasma-membranefraction of human neutrophils. A possible oxidase in the respiratoryburst. Biochem. J. 194, 599–606.

68. Yamaguchi, T., Hayakawa, T., Kaneda, M., Kakinuma, K., Yoshikawa,A. (1989) Purification and some properties of the small subunit ofcytochrome b558 from human neutrophils. J. Biol. Chem. 264, 112–118.

69. Woodman, R. C., Ruedi, J. M., Jesaitis, A. J., Okamura, N., Quinn, M. T.,Smith, R. M., Curnutte, J. T., Babior, B. M. (1991) Respiratory burstoxidase and three of four oxidase-related polypeptides are associatedwith the cytoskeleton of human neutrophils. J. Clin. Invest. 87, 1345–1351.

70. Calafat, J., Kuijpers, T. W., Janssen, H., Borregaard, N., Verhoeven,A. J., Roos, D. (1993) Evidence for small intracellular vesicles in humanblood phagocytes containing cytochrome b558 and the adhesion mole-cule CD11b/CD18. Blood 81, 3122–3129.

71. Huang, J., Hitt, N. D., Kleinberg, M. E. (1995) Stoichiometry of p22-phoxand gp91-phox in phagocyte cytochrome b558. Biochemistry 34, 16753–16757.

72. Parkos, C. A., Dinauer, M. C., Jesaitis, A. J., Orkin, S. H., Curnutte, J. T.(1989) Absence of both the 91 kD and 22 kD subunits of humanneutrophil cytochrome b in two genetic forms of chronic granulomatousdisease. Blood 73, 1416–1420.

73. Newburger, P. E., Robinson, J. M., Pryzwansky, K. B., Rosoff, P. M.,Greenberger, J. S., Tauber, A. I. (1983) Human neutrophil dysfunctionwith giant granules and defective activation of the respiratory burst.Blood 61, 1247–1257.

74. Vaissiere, C., Le Cabec, V., Maridonneau-Parini, I. (1999) NADPHoxidase is functionally assembled in specific granules during activationof human neutrophils. J. Leukoc. Biol. 65, 629–634.

75. Yu, L., Quinn, M. T., Cross, A. R., Dinauer, M. C. (1998) Gp91(phox) isthe heme-binding subunit of the superoxide-generating NADPH oxidase.Proc. Natl. Acad. Sci. USA 95, 7993–7998.

76. Hata, K., Ito, T., Takeshige, K., Sumimoto, H. (1998) Anionic amphi-phile-independent activation of the phagocyte NADPH oxidase in acell-free system by p47phox and p67phox, both in C terminally truncatedforms. Implication for regulatory Src homology 3 domain-mediated in-teractions. J. Biol. Chem. 273, 4232–4236.

77. Groemping, Y., Lapouge, K., Smerdon, S. J., Rittinger, K. (2003) Mo-lecular basis of phosphorylation-induced activation of the NADPH oxi-dase. Cell 113, 343–355.

78. Ago, T., Nunoi, H., Ito, T., Sumimoto, H. (1999) Mechanism for phos-phorylation-induced activation of the phagocyte NADPH oxidase proteinp47(phox). Triple replacement of serines 303, 304, and 328 with aspar-tates disrupts the SH3 domain-mediated intramolecular interaction inp47(phox), thereby activating the oxidase. J. Biol. Chem. 274, 33644–33653.

79. Huang, J., Kleinberg, M. E. (1999) Activation of the phagocyte NADPHoxidase protein p47(phox). Phosphorylation controls SH3 domain-depen-dent binding to p22(phox). J. Biol. Chem. 274, 19731–19737.

80. Kobayashi, T., Tsunawaki, S., Seguchi, H. (2001) Evaluation of theprocess for superoxide production by NADPH oxidase in human neutro-phils: evidence for cytoplasmic origin of superoxide. Redox Rep. 6,27–36.

81. Wilson, L., Butcher, C., Finan, P., Kellie, S. (1997) SH3 domain-mediated interactions involving the phox components of the NADPHoxidase. Inflamm. Res. 46, 265–271.

82. Crockard, A. D., Thompson, J. M., Boyd, N. A., Haughton, D. J.,McCluskey, D. R., Turner, C. P. (1997) Diagnosis and carrier detectionof chronic granulomatous disease in five families by flow cytometry. Int.Arch. Allergy Immunol. 114, 144–152.

83. Dinauer, M. C., Orkin, S. H. (1988) Chronic granulomatous disease.Molecular genetics. Hematol. Oncol. Clin. North Am. 2, 225–240.

84. Dinauer, M. C., Pierce, E. A., Erickson, R. W., Muhlebach, T. J.,Messner, H., Orkin, S. H., Seger, R. A., Curnutte, J. T. (1991) Pointmutation in the cytoplasmic domain of the neutrophil p22-phox cyto-chrome b subunit is associated with a nonfunctional NADPH oxidase andchronic granulomatous disease. Proc. Natl. Acad. Sci. USA 88, 11231–11235.

85. Porter, C. D., Parkar, M. H., Verhoeven, A. J., Levinsky, R. J., Collins,M. K., Kinnon, C. (1994) p22-phox-deficient chronic granulomatousdisease: reconstitution by retrovirus-mediated expression and identifica-tion of a biosynthetic intermediate of gp91-phox. Blood 84, 2767–2775.


86. Stasia, M. J., Bordigoni, P., Martel, C., Morel, F. (2002) A novel andunusual case of chronic granulomatous disease in a child with a homozy-gous 36-bp deletion in the CYBA gene (A22(0)) leading to the activationof a cryptic splice site in intron 4. Hum. Genet. 110, 444–450.

87. Wyche, K. E., Wang, S. S., Griendling, K. K., Dikalov, S. I., Austin, H.,Rao, S., Fink, B., Harrison, D. G., Zafari, A. M. (2004) C242T CYBApolymorphism of the NADPH oxidase is associated with reduced respi-ratory burst in human neutrophils. Hypertension 43, 1246–1251.

88. Davis, A. R., Mascolo, P. L., Bunger, P. L., Sipes, K. M., Quinn, M. T.(1998) Cloning and sequencing of the bovine flavocytochrome b subunitproteins, gp91-phox and p22-phox: comparison with other known flavo-cytochrome b sequences. J. Leukoc. Biol. 64, 114–123.

89. Harper, A. M., Chaplin, M. F., Segal, A. W. (1985) Cytochrome b-245from human neutrophils is a glycoprotein. Biochem. J. 227, 783–788.

90. Paclet, M. H., Henderson, L. M., Campion, Y., Morel, F., Dagher, M. C.(2004) Localization of Nox2 N-terminus using polyclonal antipeptideantibodies. Biochem. J. 382, 981–986.

91. Burritt, J. B., Quinn, M. T., Jutila, M. A., Bond, C. W., Jesaitis, A. J.(1995) Topological mapping of neutrophil cytochrome b epitopes withphage-display libraries. J. Biol. Chem. 270, 16974–16980.

92. Parkos, C. A., Dinauer, M. C., Walker, L. E., Allen, R. A., Jesaitis, A. J.,Orkin, S. H. (1988) Primary structure and unique expression of the22-kilodalton light chain of human neutrophil cytochrome b. Proc. Natl.Acad. Sci. USA 85, 3319–3323.

93. Burritt, J. B., Foubert, T. R., Baniulis, D., Lord, C. I., Taylor, R. M.,Mills, J. S., Baughan, T. D., Roos, D., Parkos, C. A., Jesaitis, A. J. (2003)Functional epitope on human neutrophil flavocytochrome b558. J. Im-munol. 170, 6082–6089.

94. Henderson, L. M., Chappell, J. B., Jones, O. T. (1988) Internal pHchanges associated with the activity of NADPH oxidase of human neu-trophils. Further evidence for the presence of an H� conducting chan-nel. Biochem. J. 251, 563–567.

95. Diebold, B. A., Bokoch, G. M. (2001) Molecular basis for Rac2 regulationof phagocyte NADPH oxidase. Nat. Immunol. 2, 211–215.

96. Ariga, T., Sakiyama, Y., Tomizawa, K., Imajoh-Ohmi, S., Kanegasaki, S.,Matsumoto, S. (1993) A newly recognized point mutation in the cyto-chrome b558 heavy chain gene replacing alanine57 by glutamic acid, ina patient with cytochrome b positive X-linked chronic granulomatousdisease. Eur. J. Pediatr. 152, 469–472.

97. Jendrossek, V., Ritzel, A., Neubauer, B., Heyden, S., Gahr, M. (1997) Anin-frame triplet deletion within the gp91-phox gene in an adult X-linkedchronic granulomatous disease patient with residual NADPH-oxidaseactivity. Eur. J. Haematol. 58, 78–85.

98. Rae, J., Newburger, P. E., Dinauer, M. C., Noack, D., Hopkins, P. J.,Kuruto, R., Curnutte, J. T. (1998) X-linked chronic granulomatousdisease: mutations in the CYBB gene encoding the gp91-phox componentof respiratory-burst oxidase. Am. J. Hum. Genet. 62, 1320–1331.

99. Moltyaner, Y., Geerts, W. H., Chamberlain, D. W., Heyworth, P. G.,Noack, D., Rae, J., Doyle, J. J., Downey, G. P. (2003) Underlying chronicgranulomatous disease in a patient with bronchocentric granulomatosis.Thorax 58, 1096–1098.

100. Peng, T., Lu, X., Feng, Q. (2005) NADH oxidase signaling inducescyclooxygenase-2 expression during lipopolysaccharide stimulation incardiomyocytes. FASEB J. 19, 293–295.

101. Gao, X. P., Standiford, T. J., Rahman, A., Newstead, M., Holland, S. M.,Dinauer, M. C., Liu, Q. H., Malik, A. B. (2002) Role of NADPH oxidasein the mechanism of lung neutrophil sequestration and microvesselinjury induced by Gram-negative sepsis: studies in p47phox�/� andgp91phox�/� mice. J. Immunol. 168, 3974–3982.

102. Darrah, P. A., Hondalus, M. K., Chen, Q., Ischiropoulos, H., Mosser,D. M. (2000) Cooperation between reactive oxygen and nitrogen inter-mediates in killing of Rhodococcus equi by activated macrophages. Infect.Immun. 68, 3587–3593.

103. Archer, S. L., Reeve, H. L., Michelakis, E., Puttagunta, L., Waite, R.,Nelson, D. P., Dinauer, M. C., Weir, E. K. (1999) O2 sensing is preservedin mice lacking the gp91phox subunit of NADPH oxidase. Proc. Natl.Acad. Sci. USA 96, 7944–7949.

104. Anderson, K. L., Smith, K. A., Pio, F., Torbett, B. E., Maki, R. A. (1998)Neutrophils deficient in PU.1 do not terminally differentiate or becomefunctionally competent. Blood 92, 1576–1585.

105. M’Rabet, L., Coffer, P., Zwartkruis, F., Franke, B., Segal, A. W., Koen-derman, L., Bos, J. L. (1998) Activation of the small GTPase Rap1 inhuman neutrophils. Blood 92, 2133–2140.

106. Quilliam, L. A., Mueller, H., Bohl, B. P., Prossnitz, V., Sklar, L. A., Der,C. J., Bokoch, G. M. (1991) Rap1A is a substrate for cyclic AMP-dependent protein kinase in human neutrophils. J. Immunol. 147,1628–1635.

107. Eklund, E. A., Marshall, M., Gibbs, J. B., Crean, C. D., Gabig, T. G.(1991) Resolution of a low molecular weight G protein in neutrophilcytosol required for NADPH oxidase activation and reconstitution byrecombinant Krev-1 protein. J. Biol. Chem. 266, 13964–13970.

108. Bokoch, G. M., Quilliam, L. A., Bohl, B. P., Jesaitis, A. J., Quinn, M. T.(1991) Inhibition of Rap1A binding to cytochrome b558 of NADPHoxidase by phosphorylation of Rap1A. Science 254, 1794–1796.

109. Abo, A., Pick, E., Hall, A., Totty, N., Teahan, C. G., Segal, A. W. (1991)Activation of the NADPH oxidase involves the small GTP-binding pro-tein p21rac1. Nature 353, 668–670.

110. Nakashima, S., Iwasaki, Y., Mizutani, T., Ohguchi, K., Nagata, K.,Kitajima, Y., Nozawa, Y. (1996) Differential expression of protein kinaseC isozymes and small GTP-binding proteins during HL60 cell differen-tiation by retinoic acid and cyclic AMP: relation with phospholipase D(PLD) activation. Immunobiology 196, 588–598.

111. Labadia, M. E., Bokoch, G. M., Huang, C. K. (1993) The Rap1A proteinenhances protein kinase C activity in vitro. Biochem. Biophys. Res.Commun. 195, 1321–1328.

112. Cross, A. R., Erickson, R. W., Ellis, B. A., Curnutte, J. T. (1999)Spontaneous activation of NADPH oxidase in a cell-free system: unex-pected multiple effects of magnesium ion concentrations. Biochem. J.338, 229–233.

113. Quinn, M. T., Curnutte, J. T., Parkos, C. A., Mullen, M. L., Scott, P. J.,Erickson, R. W., Jesaitis, A. J. (1992) Reconstitution of defective respi-ratory burst activity with partially purified human neutrophil cytochromeB in two genetic forms of chronic granulomatous disease: possible role ofRap1A. Blood 79, 2438–2445.

114. Bokoch, G. M., Knaus, U. G. (2003) NADPH oxidases: not just forleukocytes anymore! Trends Biochem. Sci. 28, 502–508.

115. Lambeth, J. D., Cheng, G., Arnold, R. S., Edens, W. A. (2000) Novelhomologs of gp91phox. Trends Biochem. Sci. 25, 459–461.

116. Cheng, G., Cao, Z., Xu, X., van Meir, E. G., Lambeth, J. D. (2001)Homologs of gp91phox: cloning and tissue expression of Nox3, Nox4, andNox5. Gene 269, 131–140.

117. Van Buul, J. D., Fernandez-Borja, M., Anthony, E. C., Hordijk, P. L.(2005) Expression and localization of NOX2 and NOX4 in primaryhuman endothelial cells. Antioxid. Redox Signal. 7, 308–317.

118. Ago, T., Kitazono, T., Ooboshi, H., Iyama, T., Han, Y. H., Takada, J.,Wakisaka, M., Ibayashi, S., Utsumi, H., Iida, M. (2004) Nox4 as themajor catalytic component of an endothelial NAD(P)H oxidase. Circula-tion 109, 227–233.

119. Ambasta, R. K., Kumar, P., Griendling, K. K., Schmidt, H. H., Busse, R.,Brandes, R. P. (2004) Direct interaction of the novel Nox proteins withp22phox is required for the formation of a functionally active NADPHoxidase. J. Biol. Chem. 279, 45935–45941.

120. Hilenski, L. L., Clempus, R. E., Quinn, M. T., Lambeth, J. D.,Griendling, K. K. (2004) Distinct subcellular localizations of Nox1 andNox4 in vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol.24, 677–683.

121. Dusting, G. J., Selemidis, S., Jiang, F. (2005) Mechanisms for suppress-ing NADPH oxidase in the vascular wall. Mem. Inst. Oswaldo Cruz 100(Suppl. 1), 97–103.

122. Colston, J. T., de la Rosa, S. D., Strader, J. R., Anderson, M. A.,Freeman, G. L. (2005) H2O2 activates Nox4 through PLA2-dependentarachidonic acid production in adult cardiac fibroblasts. FEBS Lett.579, 2533–2540.

123. Ueno, N., Takeya, R., Miyano, K., Kikuchi, H., Sumimoto, H. (2005) TheNADPH oxidase Nox3 constitutively produces superoxide in a p22phox-dependent manner: its regulation by oxidase organizers and activators.J. Biol. Chem. 280, 23328–23339.

124. Banfi, B., Clark, R. A., Steger, K., Krause, K. H. (2003) Two novelproteins activate superoxide generation by the NADPH oxidase NOX1.J. Biol. Chem. 278, 3510–3513.

125. Sorescu, D., Weiss, D., Lassegue, B., Clempus, R. E., Szocs, K., Sorescu,G. P., Valppu, L., Quinn, M. T., Lambeth, J. D., Vega, J. D., Taylor,W. R., Griendling, K. K. (2002) Superoxide production and expression ofNox family proteins in human atherosclerosis. Circulation 105, 1429–1435.

126. Shiose, A., Kuroda, J., Tsuruya, K., Hirai, M., Hirakata, H., Naito, S.,Hattori, M., Sakaki, Y., Sumimoto, H. (2001) A novel superoxide-producing NAD(P)H oxidase in kidney. J. Biol. Chem. 276, 1417–1423.

127. Takeya, R., Ueno, N., Kami, K., Taura, M., Kohjima, M., Izaki, T., Nunoi,H., Sumimoto, H. (2003) Novel human homologues of p47phox andp67phox participate in activation of superoxide-producing NADPH oxi-dases. J. Biol. Chem. 278, 25234–25246.

128. Li, X. J., Grunwald, D., Mathieu, J., Morel, F., Stasia, M. J. (2005)Crucial role of two potential cytosolic regions of Nox2, 191TSSTK-


TIRRS200 and 484DESQANHFAVHHDEEKD500, on NADPH oxidaseactivation. J. Biol. Chem. 280, 14962–14973.

129. Francke, U., Hsieh, C. L., Foellmer, B. E., Lomax, K. J., Malech, H. L.,Leto, T. L. (1990) Genes for two autosomal recessive forms of chronicgranulomatous disease assigned to 1q25 (NCF2) and 7q11.23 (NCF1).Am. J. Hum. Genet. 47, 483–492.

130. Rodaway, A. R., Teahan, C. G., Casimir, C. M., Segal, A. W., Bentley,D. L. (1990) Characterization of the 47-kilodalton autosomal chronicgranulomatous disease protein: tissue-specific expression and transcrip-tional control by retinoic acid. Mol. Cell. Biol. 10, 5388–5396.

131. Finan, P., Shimizu, Y., Gout, I., Hsuan, J., Truong, O., Butcher, C.,Bennett, P., Waterfield, M. D., Kellie, S. (1994) An SH3 domain andproline-rich sequence mediate an interaction between two components ofthe phagocyte NADPH oxidase complex. J. Biol. Chem. 269, 13752–13755.

132. Hiroaki, H., Ago, T., Ito, T., Sumimoto, H., Kohda, D. (2001) Solutionstructure of the PX domain, a target of the SH3 domain. Nat. Struct. Biol.8, 526–530.

133. Ponting, C. P. (1996) Novel domains in NADPH oxidase subunits, sortingnexins, and PtdIns 3-kinases: binding partners of SH3 domains? ProteinSci. 5, 2353–2357.

134. Yuzawa, S., Suzuki, N. N., Fujioka, Y., Ogura, K., Sumimoto, H., Inagaki,F. (2004) A molecular mechanism for autoinhibition of the tandem SH3domains of p47phox, the regulatory subunit of the phagocyte NADPHoxidase. Genes Cells 9, 443–456.

135. Kanai, F., Liu, H., Field, S. J., Akbary, H., Matsuo, T., Brown, G. E.,Cantley, L. C., Yaffe, M. B. (2001) The PX domains of p47phox andp40phox bind to lipid products of PI(3)K. Nat. Cell Biol. 3, 675–678.

136. Zhan, Y., Virbasius, J. V., Song, X., Pomerleau, D. P., Zhou, G. W.(2002) The p40phox and p47phox PX domains of NADPH oxidase targetcell membranes via direct and indirect recruitment by phosphoinositides.J. Biol. Chem. 277, 4512–4518.

137. Ago, T., Takeya, R., Hiroaki, H., Kuribayashi, F., Ito, T., Kohda, D.,Sumimoto, H. (2001) The PX domain as a novel phosphoinositide-binding module. Biochem. Biophys. Res. Commun. 287, 733–738.

138. Leto, T. L., Adams, A. G., de Mendez, I. (1994) Assembly of thephagocyte NADPH oxidase: binding of Src homology 3 domains toproline-rich targets. Proc. Natl. Acad. Sci. USA 91, 10650–10654.

139. Sumimoto, H., Kage, Y., Nunoi, H., Sasaki, H., Nose, T., Fukumaki, Y.,Ohno, M., Minakami, S., Takeshige, K. (1994) Role of Src homology 3domains in assembly and activation of the phagocyte NADPH oxidase.Proc. Natl. Acad. Sci. USA 91, 5345–5349.

140. Dang, P. M., Cross, A. R., Babior, B. M. (2001) Assembly of theneutrophil respiratory burst oxidase: a direct interaction betweenp67phox and cytochrome b558. Proc. Natl. Acad. Sci. USA 98, 3001–3005.

141. DeLeo, F. R., Renee, J., McCormick, S., Nakamura, M., Apicella, M.,Weiss, J. P., Nauseef, W. M. (1998) Neutrophils exposed to bacteriallipopolysaccharide upregulate NADPH oxidase assembly. J. Clin. Invest.101, 455–463.

142. Nauseef, W. M., Volpp, B. D., McCormick, S., Leidal, K. G., Clark, R. A.(1991) Assembly of the neutrophil respiratory burst oxidase. Proteinkinase C promotes cytoskeletal and membrane association of cytosolicoxidase components. J. Biol. Chem. 266, 5911–5917.

143. Reeves, E. P., Dekker, L. V., Forbes, L. V., Wientjes, F. B., Grogan, A.,Pappin, D. J., Segal, A. W. (1999) Direct interaction between p47phoxand protein kinase C: evidence for targeting of protein kinase C byp47phox in neutrophils. Biochem. J. 344 (Pt. 3), 859–866.

144. Yamamori, T., Inanami, O., Nagahata, H., Cui, Y., Kuwabara, M. (2000)Roles of p38 MAPK, PKC and PI3-K in the signaling pathways ofNADPH oxidase activation and phagocytosis in bovine polymorphonu-clear leukocytes. FEBS Lett. 467, 253–258.

145. Bey, E. A., Xu, B., Bhattacharjee, A., Oldfield, C. M., Zhao, X., Li, Q.,Subbulakshmi, V., Feldman, G. M., Wientjes, F. B., Cathcart, M. K.(2004) Protein kinase C � is required for p47phox phosphorylation andtranslocation in activated human monocytes. J. Immunol. 173, 5730–5738.

146. Dang, P. M., Fontayne, A., Hakim, J., El Benna, J., Perianin, A. (2001)Protein kinase C � phosphorylates a subset of selective sites of theNADPH oxidase component p47phox and participates in formyl peptide-mediated neutrophil respiratory burst. J. Immunol. 166, 1206–1213.

147. Fontayne, A., Dang, P. M., Gougerot-Pocidalo, M. A., El-Benna, J. (2002)Phosphorylation of p47phox sites by PKC �, �II, �, and �: effect onbinding to p22phox and on NADPH oxidase activation. Biochemistry 41,7743–7750.

148. Iwata, M., Nunoi, H., Yamazaki, H., Nakano, T., Niwa, H., Tsuruta, S.,Ohga, S., Ohmi, S., Kanegasaki, S., Matsuda, I. (1994) Homologousdinucleotide (GT or TG) deletion in Japanese patients with chronic

granulomatous disease with p47-phox deficiency. Biochem. Biophys. Res.Commun. 199, 1372–1377.

149. Gorlach, A., Lee, P. L., Roesler, J., Hopkins, P. J., Christensen, B.,Green, E. D., Chanock, S. J., Curnutte, J. T. (1997) A p47-phox pseu-dogene carries the most common mutation causing p47-phox-deficientchronic granulomatous disease. J. Clin. Invest. 100, 1907–1918.

150. Vazquez, N., Lehrnbecher, T., Chen, R., Christensen, B. L., Gallin, J. I.,Malech, H., Holland, S., Zhu, S., Chanock, S. J. (2001) Mutationalanalysis of patients with p47-phox-deficient chronic granulomatous dis-ease: the significance of recombination events between the p47-phoxgene (NCF1) and its highly homologous pseudogenes. Exp. Hematol. 29,234–243.

151. Dusi, S., Donini, M., Rossi, F. (1996) Mechanisms of NADPH oxidaseactivation: translocation of p40phox, Rac1 and Rac2 from the cytosol tothe membranes in human neutrophils lacking p47phox or p67phox.Biochem. J. 314, 409–412.

152. Sadikot, R. T., Zeng, H., Yull, F. E., Li, B., Cheng, D. S., Kernodle, D. S.,Jansen, E. D., Contag, C. H., Segal, B. H., Holland, S. M., Blackwell,T. S., Christman, J. W. (2004) p47phox deficiency impairs NF-� Bactivation and host defense in Pseudomonas pneumonia. J. Immunol.172, 1801–1808.

153. Jackson, S. H., Gallin, J. I., Holland, S. M. (1995) The p47phox mouseknock-out model of chronic granulomatous disease. J. Exp. Med. 182,751–758.

154. Sigal, N., Gorzalczany, Y., Pick, E. (2003) Two pathways of activation ofthe superoxide-generating NADPH oxidase of phagocytes in vitro—distinctive effects of inhibitors. Inflammation 27, 147–159.

155. Koshkin, V., Lotan, O., Pick, E. (1996) The cytosolic componentp47(phox) is not a sine qua non participant in the activation of NADPHoxidase but is required for optimal superoxide production. J. Biol. Chem.271, 30326–30329.

156. Leusen, J. H., Fluiter, K., Hilarius, P. M., Roos, D., Verhoeven, A. J.,Bolscher, B. G. (1995) Interactions between the cytosolic componentsp47phox and p67phox of the human neutrophil NADPH oxidase that arenot required for activation in the cell-free system. J. Biol. Chem. 270,11216–11221.

157. Uhlinger, D. J., Taylor, K. L., Lambeth, J. D. (1994) p67-phox enhancesthe binding of p47-phox to the human neutrophil respiratory burstoxidase complex. J. Biol. Chem. 269, 22095–22098.

158. Kleinberg, M. E., Mital, D., Rotrosen, D., Malech, H. L. (1992) Charac-terization of a phagocyte cytochrome b558 91-kilodalton subunit func-tional domain: identification of peptide sequence and amino acids es-sential for activity. Biochemistry 31, 2686–2690.

159. Fuchs, A., Dagher, M. C., Faure, J., Vignais, P. V. (1996) Topologicalorganization of the cytosolic activating complex of the superoxide-gen-erating NADPH-oxidase. Pinpointing the sites of interaction betweenp47phoz, p67phox and p40phox using the two-hybrid system. Biochim.Biophys. Acta 1312, 39–47.

160. Nakamura, R., Sumimoto, H., Mizuki, K., Hata, K., Ago, T., Kitajima, S.,Takeshige, K., Sakaki, Y., Ito, T. (1998) The PC motif: a novel andevolutionarily conserved sequence involved in interaction betweenp40phox and p67phox, SH3 domain-containing cytosolic factors of thephagocyte NADPH oxidase. Eur. J. Biochem. 251, 583–589.

161. Wientjes, F. B., Panayotou, G., Reeves, E., Segal, A. W. (1996) Inter-actions between cytosolic components of the NADPH oxidase: p40phoxinteracts with both p67phox and p47phox. Biochem. J. 317, 919–924.

162. Dusi, S., Rossi, F. (1993) Activation of NADPH oxidase of humanneutrophils involves the phosphorylation and the translocation of cyto-solic p67phox. Biochem. J. 296, 367–371.

163. Smith, R. M., Connor, J. A., Chen, L. M., Babior, B. M. (1996) Thecytosolic subunit p67phox contains an NADPH-binding site that partic-ipates in catalysis by the leukocyte NADPH oxidase. J. Clin. Invest. 98,977–983.

164. Freeman, J. L., Abo, A., Lambeth, J. D. (1996) Rac “insert region” is anovel effector region that is implicated in the activation of NADPHoxidase, but not PAK65. J. Biol. Chem. 271, 19794–19801.

165. Dang, P. M., Babior, B. M., Smith, R. M. (1999) NADPH dehydrogenaseactivity of p67phox, a cytosolic subunit of the leukocyte NADPH oxidase.Biochemistry 38, 5746–5753.

166. Zicha, D., Dunn, G. A., Segal, A. W. (1997) Deficiency of p67phox,p47phox or gp91phox in chronic granulomatous disease does not impairleucocyte chemotaxis or motility. Br. J. Haematol. 96, 543–550.

167. Polack, B., Vergnaud, S., Paclet, M. H., Lamotte, D., Toussaint, B.,Morel, F. (2000) Protein delivery by Pseudomonas type III secretionsystem: ex vivo complementation of p67(phox)-deficient chronic granu-lomatous disease. Biochem. Biophys. Res. Commun. 275, 854–858.

168. Noack, D., Rae, J., Cross, A. R., Munoz, J., Salmen, S., Mendoza, J. A.,Rossi, N., Curnutte, J. T., Heyworth, P. G. (1999) Autosomal recessive


chronic granulomatous disease caused by novel mutations in NCF-2, thegene encoding the p67-phox component of phagocyte NADPH oxidase.Hum. Genet. 105, 460–467.

169. Ahlin, A., De, B. M., Roos, D., Leusen, J., Smith, C. I., Sundin, U.,Rabbani, H., Palmblad, J., Elinder, G. (1995) Prevalence, genetics andclinical presentation of chronic granulomatous disease in Sweden. ActaPaediatr. 84, 1386–1394.

170. Tanugi-Cholley, L. C., Issartel, J. P., Lunardi, J., Freycon, F., Morel, F.,Vignais, P. V. (1995) A mutation located at the 5� splice junctionsequence of intron 3 in the p67phox gene causes the lack of p67phoxmRNA in a patient with chronic granulomatous disease. Blood 85,242–249.

171. Cross, A. R. (2000) p40(phox) participates in the activation of NADPHoxidase by increasing the affinity of p47(phox) for flavocytochromeb(558). Biochem. J. 349, 113–117.

172. Sathyamoorthy, M., de Mendez, I., Adams, A. G., Leto, T. L. (1997)p40(phox) down-regulates NADPH oxidase activity through interactionswith its SH3 domain. J. Biol. Chem. 272, 9141–9146.

173. Someya, A., Nunoi, H., Hasebe, T., Nagaoka, I. (1999) Phosphorylationof p40-phox during activation of neutrophil NADPH oxidase. J. Leukoc.Biol. 66, 851–857.

174. Vergnaud, S., Paclet, M. H., El Benna, J., Pocidalo, M. A., Morel, F.(2000) Complementation of NADPH oxidase in p67-phox-deficient CGDpatients p67-phox/p40-phox interaction. Eur. J. Biochem. 267, 1059–1067.

175. Wientjes, F. B., Hsuan, J. J., Totty, N. F., Segal, A. W. (1993) p40phox,a third cytosolic component of the activation complex of the NADPHoxidase to contain src homology 3 domains. Biochem. J. 296, 557–561.

176. Zhan, S., Vazquez, N., Zhan, S., Wientjes, F. B., Budarf, M. L., Schrock,E., Ried, T., Green, E. D., Chanock, S. J. (1996) Genomic structure,chromosomal localization, start of transcription, and tissue expression ofthe human p40-phox, a new component of the nicotinamide adeninedinucleotide phosphate-oxidase complex. Blood 88, 2714–2721.

177. Tsunawaki, S., Mizunari, H., Nagata, M., Tatsuzawa, O., Kuratsuji, T.(1994) A novel cytosolic component, p40phox, of respiratory burst oxi-dase associates with p67phox and is absent in patients with chronicgranulomatous disease who lack p67phox. Biochem. Biophys. Res. Com-mun. 199, 1378–1387.

178. Tsunawaki, S., Kagara, S., Yoshikawa, K., Yoshida, L. S., Kuratsuji, T.,Namiki, H. (1996) Involvement of p40phox in activation of phagocyteNADPH oxidase through association of its carboxyl-terminal, but not itsamino-terminal, with p67phox. J. Exp. Med. 184, 893–902.

179. Fuchs, A., Dagher, M. C., Vignais, P. V. (1995) Mapping the domains ofinteraction of p40phox with both p47phox and p67phox of the neutrophiloxidase complex using the two-hybrid system. J. Biol. Chem. 270,5695–5697.

180. Fuchs, A., Bouin, A. P., Rabilloud, T., Vignais, P. V. (1997) The 40-kDacomponent of the phagocyte NADPH oxidase (p40phox) is phosphory-lated during activation in differentiated HL60 cells. Eur. J. Biochem.249, 531–539.

181. Bouin, A. P., Grandvaux, N., Vignais, P. V., Fuchs, A. (1998) p40(phox)is phosphorylated on threonine 154 and serine 315 during activation ofthe phagocyte NADPH oxidase. Implication of a protein kinase C-typekinase in the phosphorylation process. J. Biol. Chem. 273, 30097–30103.

182. Knaus, U. G., Heyworth, P. G., Evans, T., Curnutte, J. T., Bokoch, G. M.(1991) Regulation of phagocyte oxygen radical production by the GTP-binding protein Rac 2. Science 254, 1512–1515.

183. Knaus, U. G., Heyworth, P. G., Kinsella, B. T., Curnutte, J. T., Bokoch,G. M. (1992) Purification and characterization of Rac 2. A cytosolicGTP-binding protein that regulates human neutrophil NADPH oxidase.J. Biol. Chem. 267, 23575–23582.

184. Abo, A., Webb, M. R., Grogan, A., Segal, A. W. (1994) Activation ofNADPH oxidase involves the dissociation of p21rac from its inhibitoryGDP/GTP exchange protein (rhoGDI) followed by its translocation to theplasma membrane. Biochem. J. 298 (Pt. 3), 585–591.

185. Quinn, M. T., Evans, T., Loetterle, L. R., Jesaitis, A. J., Bokoch, G. M.(1993) Translocation of Rac correlates with NADPH oxidase activation.Evidence for equimolar translocation of oxidase components. J. Biol.Chem. 268, 20983–20987.

186. Prigmore, E., Ahmed, S., Best, A., Kozma, R., Manser, E., Segal, A. W.,Lim, L. (1995) A 68-kDa kinase and NADPH oxidase componentp67phox are targets for Cdc42Hs and Rac1 in neutrophils. J. Biol. Chem.270, 10717–10722.

187. Dusi, S., Donini, M., Rossi, F. (1995) Mechanisms of NADPH oxidaseactivation in human neutrophils: p67phox is required for the transloca-tion of Rac 1 but not of Rac 2 from cytosol to the membranes. Biochem.J. 308, 991–994.

188. Ahmed, S., Prigmore, E., Govind, S., Veryard, C., Kozma, R., Wientjes,F. B., Segal, A. W., Lim, L. (1998) Cryptic Rac-binding andp21(Cdc42Hs/Rac)-activated kinase phosphorylation sites of NADPHoxidase component p67(phox). J. Biol. Chem. 273, 15693-15701.

189. Rinckel, L. A., Faris, S. L., Hitt, N. D., Kleinberg, M. E. (1999) Rac1disrupts p67phox/p40phox binding: a novel role for Rac in NADPHoxidase activation. Biochem. Biophys. Res. Commun. 263, 118–122.

190. Freeman, J. L., Kreck, M. L., Uhlinger, D. J., Lambeth, J. D. (1994) Raseffector-homologue region on Rac regulates protein associations in theneutrophil respiratory burst oxidase complex. Biochemistry 33, 13431–13435.

191. Filippi, M. D., Harris, C. E., Meller, J., Gu, Y., Zheng, Y., Williams,D. A. (2004) Localization of Rac2 via the C terminus and aspartic acid150 specifies superoxide generation, actin polarity and chemotaxis inneutrophils. Nat. Immunol. 5, 744–751.

192. Yang, F. C., Kapur, R., King, A. J., Tao, W., Kim, C., Borneo, J., Breese,R., Marshall, M., Dinauer, M. C., Williams, D. A. (2000) Rac2 stimulatesAkt activation affecting BAD/Bcl-XL expression while mediating sur-vival and actin function in primary mast cells. Immunity 12, 557–568.

193. Gu, Y., Williams, D. A. (2002) RAC2 GTPase deficiency and myeloidcell dysfunction in human and mouse. J. Pediatr. Hematol. Oncol. 24,791–794.

194. Yamauchi, A., Kim, C., Li, S., Marchal, C. C., Towe, J., Atkinson, S. J.,Dinauer, M. C. (2004) Rac2-deficient murine macrophages have selec-tive defects in superoxide production and phagocytosis of opsonizedparticles. J. Immunol. 173, 5971–5979.

195. Yamauchi, A., Marchal, C. C., Molitoris, J., Pech, N., Knaus, U., Towe,J., Atkinson, S. J., Dinauer, M. C. (2005) Rac GTPase isoform-specificregulation of NADPH oxidase and chemotaxis in murine neutrophils invivo. Role of the C-terminal polybasic domain. J. Biol. Chem. 280,953–964.

196. Abdel-Latif, D., Steward, M., Lacy, P. (2005) Neutrophil primary granulerelease and maximal superoxide generation depend on Rac2 in a com-mon signaling pathway. Can. J. Physiol. Pharmacol. 83, 69–75.

197. Bokoch, G. M. (2005) Regulation of innate immunity by Rho GTPases.Trends Cell Biol. 15, 163–171.

198. Carstanjen, D., Yamauchi, A., Koornneef, A., Zang, H., Filippi, M. D.,Harris, C., Towe, J., Atkinson, S., Zheng, Y., Dinauer, M. C., Williams,D. A. (2005) Rac2 regulates neutrophil chemotaxis, superoxide produc-tion, and myeloid colony formation through multiple distinct effectorpathways. J. Immunol. 174, 4613–4620.

199. Kim, C., Dinauer, M. C. (2001) Rac2 is an essential regulator of neu-trophil nicotinamide adenine dinucleotide phosphate oxidase activationin response to specific signaling pathways. J. Immunol. 166, 1223–1232.

200. Ambruso, D. R., Knall, C., Abell, A. N., Panepinto, J., Kurkchubasche,A., Thurman, G., Gonzalez-Aller, C., Hiester, A., deBoer, M., Harbeck,R. J., Oyer, R., Johnson, G. L., Roos, D. (2000) Human neutrophilimmunodeficiency syndrome is associated with an inhibitory Rac2 mu-tation. Proc. Natl. Acad. Sci. USA 97, 4654–4659.

201. Diebold, B. A., Fowler, B., Lu, J., Dinauer, M. C., Bokoch, G. M. (2004)Antagonistic cross-talk between Rac and Cdc42 GTPases regulatesgeneration of reactive oxygen species. J. Biol. Chem. 279, 28136–28142.

202. Kwong, C. H., Malech, H. L., Rotrosen, D., Leto, T. L. (1993) Regulationof the human neutrophil NADPH oxidase by rho-related G-proteins.Biochemistry 32, 5711–5717.

203. Kwong, C. H., Adams, A. G., Leto, T. L. (1995) Characterization of theeffector-specifying domain of Rac involved in NADPH oxidase activa-tion. J. Biol. Chem. 270, 19868–19872.

204. Shalom-Barak, T., Knaus, U. G. (2002) A p21-activated kinase-con-trolled metabolic switch up-regulates phagocyte NADPH oxidase. J. Biol.Chem. 277, 40659–40665.

205. Dib, K., Melander, F., Axelsson, L., Dagher, M. C., Aspenstrom, P.,Andersson, T. (2003) Down-regulation of Rac activity during � 2 inte-grin-mediated adhesion of human neutrophils. J. Biol. Chem. 278,24181–24188.

206. Silliman, C. C. (1999) Transfusion-related acute lung injury. Transfus.Med. Rev. 13, 177–186.

207. Silliman, C. C., Ambruso, D. R., Boshkov, L. K. (2004) Transfusion-related acute lung injury. Blood 105, 2266–2273.

208. Gao, X. P., Standiford, T. J., Rahman, A., Newstead, M., Holland, S. M.,Dinauer, M. C., Liu, Q. H., Malik, A. B. (2002) Role of NADPH oxidasein the mechanism of lung neutrophil sequestration and microvesselinjury induced by Gram-negative sepsis: studies in p47phox�/� andgp91phox�/� mice. J. Immunol. 168, 3974–3982.

209. Silliman, C. C., Clay, K. L., Thurman, G. W., Johnson, C. A., Ambruso,D. R. (1994) Partial characterization of lipids that develop during the


routine storage of blood and prime the neutrophil NADPH oxidase.J. Lab. Clin. Med. 124, 684–694.

210. Woo, C. H., Yoo, M. H., You, H. J., Cho, S. H., Mun, Y. C., Seong, C. M.,Kim, J. H. (2003) Transepithelial migration of neutrophils in response toleukotriene B4 is mediated by a reactive oxygen species-extracellularsignal-regulated kinase-linked cascade. J. Immunol. 170, 6273–6279.

211. Wyman, T. H., Dinarello, C. A., Banerjee, A., Gamboni-Robertson, F.,Hiester, A. A., England, K. M., Kelher, M., Silliman, C. C. (2002)Physiological levels of interleukin-18 stimulate multiple neutrophil func-tions through p38 MAP kinase activation. J. Leukoc. Biol. 72, 401–409.

212. Lapouge, K., Smith, S. J., Groemping, Y., Rittinger, K. (2002) Architec-ture of the p40–p47-p67phox complex in the resting state of the NADPHoxidase. A central role for p67phox. J. Biol. Chem. 277, 10121–10128.

213. Leusen, J. H., Verhoeven, A. J., Roos, D. (1996) Interactions between thecomponents of the human NADPH oxidase: a review about the intriguesin the phox family. Front. Biosci. 1, d72–d90.

214. Dang, P. M., Cross, A. R., Quinn, M. T., Babior, B. M. (2002) Assemblyof the neutrophil respiratory burst oxidase: a direct interaction betweenp67phox and cytochrome b558 II. Proc. Natl. Acad. Sci. USA 99,4262–4265.

215. Brown, G. E., Stewart, M. Q., Liu, H., Ha, V. L., Yaffe, M. B. (2003) Anovel assay system implicates PtdIns(3,4)P(2), PtdIns(3)P, and PKC � inintracellular production of reactive oxygen species by the NADPH oxi-dase. Mol. Cell 11, 35–47.

216. Babior, B. M. (1994) Activation of the respiratory burst oxidase. Environ.Health Perspect. 102 (Suppl. 10), 53–56.

217. Wientjes, F. B., Segal, A. W., Hartwig, J. H. (1997) Immunoelectronmicroscopy shows a clustered distribution of NADPH oxidase compo-nents in the human neutrophil plasma membrane. J. Leukoc. Biol. 61,303–312.

218. El Benna, J., Dang, P. M., Andrieu, V., Vergnaud, S., Dewas, C., Cachia,O., Fay, M., Morel, F., Chollet-Martin, S., Hakim, J., Gougerot-Pocidalo,M. A. (1999) p40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: acomparative study with p67phox and p47phox. J. Leukoc. Biol. 66,1014–1020.

219. Yan, S. R., Fumagalli, L., Dusi, S., Berton, G. (1995) Tumor necrosisfactor triggers redistribution to a Triton X-100-insoluble, cytoskeletalfraction of � 2 integrins, NADPH oxidase components, tyrosine phos-phorylated proteins, and the protein tyrosine kinase p58fgr in humanneutrophils adherent to fibrinogen. J. Leukoc. Biol. 58, 595–606.

220. Borregaard, N. (1985) The respiratory burst of phagocytosis: biochemis-try and subcellular localization. Immunol. Lett. 11, 165–171.

221. Dewald, B., Thelen, M., Baggiolini, M. (1988) Two transduction se-quences are necessary for neutrophil activation by receptor agonists.J. Biol. Chem. 263, 16179–16184.

222. McPhail, L. C., Clayton, C. C., Snyderman, R. (1984) The NADPHoxidase of human polymorphonuclear leukocytes. Evidence for regulationby multiple signals. J. Biol. Chem. 259, 5768–5775.

223. Palmblad, J., Gyllenhammar, H., Lindgren, J. A., Malmsten, C. L. (1984)Effects of leukotrienes and f-Met-Leu-Phe on oxidative metabolism ofneutrophils and eosinophils. J. Immunol. 132, 3041–3045.

224. Mansfield, P. J., Hinkovska-Galcheva, V., Shayman, J. A., Boxer, L. A.(2002) Granulocyte colony-stimulating factor primes NADPH oxidase inneutrophils through translocation of cytochrome b(558) by gelatinase-granule release. J. Lab. Clin. Med. 140, 9–16.

225. Gay, J. C. (1990) Priming of neutrophil oxidative responses by platelet-activating factor. J. Lipid Mediat. 2 (Suppl.), S161–S175.

226. Vercellotti, G. M., Yin, H. Q., Gustafson, K. S., Nelson, R. D., Jacob,H. S. (1988) Platelet-activating factor primes neutrophil responses toagonists: role in promoting neutrophil-mediated endothelial damage.Blood 71, 1100–1107.

227. Kitchen, E., Rossi, A. G., Condliffe, A. M., Haslett, C., Chilvers, E. R.(1996) Demonstration of reversible priming of human neutrophils usingplatelet-activating factor. Blood 88, 4330–4337.

228. Daniels, R. H., Finnen, M. J., Hill, M. E., Lackie, J. M. (1992) Recom-binant human monocyte IL-8 primes NADPH-oxidase and phospholipaseA2 activation in human neutrophils. Immunology 75, 157–163.

229. Ferrante, A. (1992) Activation of neutrophils by interleukins-1 and -2and tumor necrosis factors. Immunol. Ser. 57, 417–436.

230. Luchi, M., Munford, R. S. (1993) Binding, internalization, and deacyla-tion of bacterial lipopolysaccharide by human neutrophils. J. Immunol.151, 959–969.

231. Dang, P. M., Dewas, C., Gaudry, M., Fay, M., Pedruzzi, E., Gougerot-Pocidalo, M. A., El Benna, J. (1999) Priming of human neutrophilrespiratory burst by granulocyte/macrophage colony-stimulating factor(GM-CSF) involves partial phosphorylation of p47(phox). J. Biol. Chem.274, 20704–20708.

232. Levy, R., Dana, R., Leto, T. L., Malech, H. L. (1994) The requirement ofp47 phosphorylation for activation of NADPH oxidase by opsonizedzymosan in human neutrophils. Biochim. Biophys. Acta 1220, 253–260.

233. Dewas, C., Dang, P. M., Gougerot-Pocidalo, M. A., El-Benna, J. (2003)TNF-� induces phosphorylation of p47(phox) in human neutrophils:partial phosphorylation of p47phox is a common event of priming ofhuman neutrophils by TNF-� and granulocyte-macrophage colony-stim-ulating factor. J. Immunol. 171, 4392–4398.

234. Brown, G. E., Stewart, M. Q., Bissonnette, S. A., Elia, A. E., Wilker, E.,Yaffe, M. B. (2004) Distinct ligand-dependent roles for p38 MAPK inpriming and activation of the neutrophil NADPH oxidase. J. Biol. Chem.279, 27059–27068.

235. El Benna, J., Hayem, G., Dang, P. M., Fay, M., Chollet-Martin, S., Elbim,C., Meyer, O., Gougerot-Pocidalo, M. A. (2002) NADPH oxidase primingand p47phox phosphorylation in neutrophils from synovial fluid of pa-tients with rheumatoid arthritis and spondylarthropathy. Inflammation26, 273–278.

236. Sheppard, F. R., Moore, E. E., McLaughlin, N., Kelher, M., Johnson,J. L., Silliman, C. C. (2005) Clinically relevant osmolar stress inhibitspriming-induced PMN NADPH oxidase subunit translocation. J. Trauma58, 752–757.

237. Alvarez-Maqueda, M., El Bekay, R., Monteseirin, J., Alba, G., Chacon,P., Vega, A., Santa, M. C., Tejedo, J. R., Martin-Nieto, J., Bedoya, F. J.,Pintado, E., Sobrino, F. (2004) Homocysteine enhances superoxide anionrelease and NADPH oxidase assembly by human neutrophils. Effects onMAPK activation and neutrophil migration. Atherosclerosis 172, 229–238.

238. El Bekay, R., Alvarez, M., Monteseirin, J., Alba, G., Chacon, P., Vega,A., Martin-Nieto, J., Jimenez, J., Pintado, E., Bedoya, F. J., Sobrino, F.(2003) Oxidative stress is a critical mediator of the angiotensin II signalin human neutrophils: involvement of mitogen-activated protein kinase,calcineurin, and the transcription factor NF-�B. Blood 102, 662–671.

239. Touyz, R. M., Chen, X., Tabet, F., Yao, G., He, G., Quinn, M. T., Pagano,P. J., Schiffrin, E. L. (2002) Expression of a functionally activegp91phox-containing neutrophil-type NAD(P)H oxidase in smooth mus-cle cells from human resistance arteries: regulation by angiotensin II.Circ. Res. 90, 1205–1213.

240. Utsumi, T., Klostergaard, J., Akimaru, K., Edashige, K., Sato, E. F.,Utsumi, K. (1992) Modulation of TNF-�-priming and stimulation-depen-dent superoxide generation in human neutrophils by protein kinaseinhibitors. Arch. Biochem. Biophys. 294, 271–278.

241. Silliman, C. C., Elzi, D. J., Ambruso, D. R., Musters, R. J., Hamiel, C.,Harbeck, R. J., Paterson, A. J., Bjornsen, A. J., Wyman, T. H., Kelher,M., England, K. M., Laughlin-Malaxecheberria, N., Barnett, C. C., Ai-boshi, J., Bannerjee, A. (2003) Lysophosphatidylcholines prime theNADPH oxidase and stimulate multiple neutrophil functions throughchanges in cytosolic calcium. J. Leukoc. Biol. 73, 511–524.

242. Bender, J. G., McPhail, L. C., Van Epps, D. E. (1983) Exposure of humanneutrophils to chemotactic factors potentiates activation of the respira-tory burst enzyme. J. Immunol. 130, 2316–2323.

243. McLeish, K. R., Knall, C., Ward, R. A., Gerwins, P., Coxon, P. Y., Klein,J. B., Johnson, G. L. (1998) Activation of mitogen-activated proteinkinase cascades during priming of human neutrophils by TNF-� andGM-CSF. J. Leukoc. Biol. 64, 537–545.

244. Dang, P. M., Morel, F., Gougerot-Pocidalo, M. A., Benna, J. E. (2003)Phosphorylation of the NADPH oxidase component p67(phox) by ERK2and P38MAPK: selectivity of phosphorylated sites and existence of anintramolecular regulatory domain in the tetratricopeptide-rich region.Biochemistry 42, 4520–4526.

245. Dusi, S., Donini, M., Rossi, F. (1994) Tyrosine phosphorylation andactivation of NADPH oxidase in human neutrophils: a possible role forMAP kinases and for a 75 kDa protein. Biochem. J. 304, 243–250.

246. Fu, H., Bylund, J., Karlsson, A., Pellme, S., Dahlgren, C. (2004) Themechanism for activation of the neutrophil NADPH-oxidase by thepeptides formyl-Met-Leu-Phe and Trp-Lys-Tyr-Met-Val-Met differs fromthat for interleukin-8. Immunology 112, 201–210.

247. Kelher, M. R., Ambruso, D. R., Elzi, D. J., Anderson, S. M., Paterson,A. J., Thurman, G. W., Silliman, C. C. (2003) formyl-Met-Leu-Pheinduces calcium-dependent tyrosine phosphorylation of Rel-1 in neutro-phils. Cell Calcium 34, 445–455.

248. Torres, M., Forman, H. J. (1999) Activation of several MAP kinases uponstimulation of rat alveolar macrophages: role of the NADPH oxidase.Arch. Biochem. Biophys. 366, 231–239.

249. Yamamori, T., Inanami, O., Sumimoto, H., Akasaki, T., Nagahata, H.,Kuwabara, M. (2002) Relationship between p38 mitogen-activated pro-tein kinase and small GTPase Rac for the activation of NADPH oxidasein bovine neutrophils. Biochem. Biophys. Res. Commun. 293, 1571–1578.


250. Ward, R. A., Nakamura, M., McLeish, K. R. (2000) Priming of theneutrophil respiratory burst involves p38 mitogen-activated protein ki-nase-dependent exocytosis of flavocytochrome b558-containing granules.J. Biol. Chem. 275, 36713–36719.

251. Kettritz, R., Schreiber, A., Luft, F. C., Haller, H. (2001) Role of mitogen-activated protein kinases in activation of human neutrophils by antineu-trophil cytoplasmic antibodies. J. Am. Soc. Nephrol. 12, 37–46.

252. Elzi, D. J., Bjornsen, A. J., MacKenzie, T., Wyman, T. H., Silliman, C. C.(2001) Ionomycin causes activation of p38 and p42/44 mitogen-activatedprotein kinases in human neutrophils. Am. J. Physiol. Cell Physiol. 281,C350–C360.

253. Werz, O., Klemm, J., Samuelsson, B., Radmark, O. (2000) 5-Lipoxygen-ase is phosphorylated by p38 kinase-dependent MAPKAP kinases. Proc.Natl. Acad. Sci. USA 97, 5261–5266.

254. Mollapour, E., Linch, D. C., Roberts, P. J. (2001) Activation and primingof neutrophil nicotinamide adenine dinucleotide phosphate oxidase andphospholipase A(2) are dissociated by inhibitors of the kinasesp42(ERK2) and p38(SAPK) and by methyl arachidonyl fluorophospho-nate, the dual inhibitor of cytosolic and calcium-independent phospho-lipase A(2). Blood 97, 2469–2477.

255. Werz, O., Burkert, E., Samuelsson, B., Radmark, O., Steinhilber, D.(2002) Activation of 5-lipoxygenase by cell stress is calcium independentin human polymorphonuclear leukocytes. Blood 99, 1044–1052.

256. Burkert, E., Szellas, D., Radmark, O., Steinhilber, D., Werz, O. (2003)Cell type-dependent activation of 5-lipoxygenase by arachidonic acid.J. Leukoc. Biol. 73, 191–200.

257. Bylund, J., Bjorstad, A., Granfeldt, D., Karlsson, A., Woschnagg, C.,Dahlgren, C. (2003) Reactivation of formyl peptide receptors triggers theneutrophil NADPH-oxidase but not a transient rise in intracellularcalcium. J. Biol. Chem. 278, 30578–30586.

258. Dennis, E. A., Rhee, S. G., Billah, M. M., Hannun, Y. A. (1991) Role ofphospholipase in generating lipid second messengers in signal transduc-tion. FASEB J. 5, 2068–2077.

259. Silliman, C. C., Dickey, W. O., Paterson, A. J., Thurman, G. W., Clay,K. L., Johnson, C. A., Ambruso, D. R. (1996) Analysis of the primingactivity of lipids generated during routine storage of platelet concen-trates. Transfusion 36, 133–139.

260. Muller, J., Petkovic, M., Schiller, J., Arnold, K., Reichl, S., Arnhold, J.(2002) Effects of lysophospholipids on the generation of reactive oxygenspecies by fMLP- and PMA-stimulated human neutrophils. Luminescence17, 141–149.

261. Dana, R., Leto, T. L., Malech, H. L., Levy, R. (1998) Essential require-ment of cytosolic phospholipase A2 for activation of the phagocyteNADPH oxidase. J. Biol. Chem. 273, 441–445.

262. Henderson, W. R., Klebanoff, S. J. (1983) Leukotriene production andinactivation by normal, chronic granulomatous disease and myeloperox-idase-deficient neutrophils. J. Biol. Chem. 258, 13522–13527.

263. Daniels, I., Lindsay, M. A., Keany, C. I., Burden, R. P., Fletcher, J.,Haynes, A. P. (1998) Role of arachidonic acid and its metabolites in thepriming of NADPH oxidase in human polymorphonuclear leukocytes byperitoneal dialysis effluent. Clin. Diagn. Lab. Immunol. 5, 683–689.

264. Smolen, J. E., Stoehr, S. J. (1986) Guanine nucleotides reduce the freecalcium requirement for secretion of granule constituents from perme-abilized human neutrophils. Biochim. Biophys. Acta 889, 171–178.

265. Nasmith, P. E., Mills, G. B., Grinstein, S. (1989) Guanine nucleotidesinduce tyrosine phosphorylation and activation of the respiratory burst inneutrophils. Biochem. J. 257, 893–897.

266. Klinker, J. F., Wenzel-Seifert, K., Seifert, R. (1996) G-protein-coupledreceptors in HL-60 human leukemia cells. Gen. Pharmacol. 27, 33–54.

267. Keil, M. L., Solomon, N. L., Lodhi, I. J., Stone, K. C., Jesaitis, A. J.,Chang, P. S., Linderman, J. J., Omann, G. M. (2003) Priming-inducedlocalization of G(i�2) in high density membrane microdomains. Biochem.Biophys. Res. Commun. 301, 862–872.

268. Capodici, C., Pillinger, M. H., Han, G., Philips, M. R., Weissmann, G.(1998) Integrin-dependent homotypic adhesion of neutrophils. Arachi-donic acid activates Raf-1/Mek/Erk via a 5-lipoxygenase-dependentpathway. J. Clin. Invest. 102, 165–175.

269. Hsu, M. F., Lu, M. C., Tsao, L. T., Kuan, Y. H., Chen, C. C., Wang, J. P.(2004) Mechanisms of the influence of magnolol on eicosanoid metabo-lism in neutrophils. Biochem. Pharmacol. 67, 831–840.

270. Abdel-Latif, D., Steward, M., Macdonald, D. L., Francis, G. A., Dinauer,M. C., Lacy, P. (2004) Rac2 is critical for neutrophil primary granuleexocytosis. Blood 104, 832–839.

271. Heyworth, P. G., Badwey, J. A. (1990) Protein phosphorylation associ-ated with the stimulation of neutrophils. Modulation of superoxide pro-duction by protein kinase C and calcium. J. Bioenerg. Biomembr. 22,1–26.

272. Nixon, J. B., McPhail, L. C. (1999) Protein kinase C (PKC) isoformstranslocate to Triton-insoluble fractions in stimulated human neutro-phils: correlation of conventional PKC with activation of NADPH oxi-dase. J. Immunol. 163, 4574–4582.

273. Andrews, P. C., Babior, B. M. (1984) Phosphorylation of cytosolic pro-teins by resting and activated human neutrophils. Blood 64, 883–890.

274. Khalfi, F., Gressier, B., Brunet, C., Dine, T., Luyckx, M., Cazin, M.,Cazin, J. C. (1996) Involvement of the extracellular calcium in therelease of elastase and the human neutrophils oxidative burst. Cell. Mol.Biol. (Noisy-le-grand) 42, 1211–1218.

275. Elzi, D. J., Bjornsen, A. J., MacKenzie, T., Wyman, T. H., Silliman, C. C.(2001) Ionomycin causes activation of p38 and p42/44 mitogen-activatedprotein kinases in human neutrophils. Am. J. Physiol. Cell Physiol. 281,C350–C360.

276. Ferby, I., Waga, I., Kume, K., Sakanaka, C., Shimizu, T. (1996) PAF-induced MAPK activation is inhibited by wortmannin in neutrophils andmacrophages. Adv. Exp. Med. Biol. 416, 321–326.

277. Naccache, P. H., Showell, H. J., Becker, E. L., Sha’afi, R. I. (1979) Thechemotactic factors induced movement of calcium and sodium acrossrabbit neutrophil membranes: effect of densensitization to cytochalasinB. J. Cell. Physiol. 100, 239–250.

278. Naccache, P. H., Showell, H. J., Becker, E. L., Sha’afi, R. I. (1979)Involvement of membrane calcium in the response of rabbit neutrophilsto chemotactic factors as evidenced by the fluorescence of chlorotetra-cycline. J. Cell Biol. 83, 179–186.

279. Naccache, P. H., Therrien, S., Caon, A. C., Liao, N., Gilbert, C., McColl,S. R. (1989) Chemoattractant-induced cytoplasmic pH changes andcytoskeletal reorganization in human neutrophils. Relationship to thestimulated calcium transients and oxidative burst. J. Immunol. 142,2438–2444.

280. Jankowski, A., Grinstein, S. (1999) A noninvasive fluorimetric procedurefor measurement of membrane potential. Quantification of the NADPHoxidase-induced depolarization in activated neutrophils. J. Biol. Chem.274, 26098–26104.

281. Dahlgren, C. (1989) The calcium ionophore ionomycin can prime, but notactivate, the reactive oxygen generating system in differentiated HL-60cells. J. Leukoc. Biol. 46, 15–24.

282. Liu, L., Harbecke, O., Elwing, H., Follin, P., Karlsson, A., Dahlgren, C.(1998) Desensitization of formyl peptide receptors is abolished in cal-cium ionophore-primed neutrophils: an association of the ligand-receptorcomplex to the cytoskeleton is not required for a rapid termination of theNADPH-oxidase response. J. Immunol. 160, 2463–2468.

283. Quinn, M. T., Parkos, C. A., Jesaitis, A. J. (1989) The lateral organizationof components of the membrane skeleton and superoxide generation inthe plasma membrane of stimulated human neutrophils. Biochim. Bio-phys. Acta 987, 83–94.

284. Ridley, A. J., Paterson, H. F., Johnston, C. L., Diekmann, D., Hall, A.(1992) The small GTP-binding protein Rac regulates growth factor-induced membrane ruffling. Cell 70, 401–410.

285. Morimatsu, T., Kawagoshi, A., Yoshida, K., Tamura, M. (1997) Actinenhances the activation of human neutrophil NADPH oxidase in acell-free system. Biochem. Biophys. Res. Commun. 230, 206–210.

286. Chung, C. Y., Lee, S., Briscoe, C., Ellsworth, C., Firtel, R. A. (2000) Roleof Rac in controlling the actin cytoskeleton and chemotaxis in motilecells. Proc. Natl. Acad. Sci. USA 97, 5225–5230.

287. Suzuki, K., Nishihata, J., Arai, Y., Honma, N., Yamamoto, K., Irimura,T., Toyoshima, S. (1995) Molecular cloning of a novel actin-bindingprotein, p57, with a WD repeat and a leucine zipper motif. FEBS Lett.364, 283–288.

288. de Hostos, E. L., Bradtke, B., Lottspeich, F., Guggenheim, R., Gerisch,G. (1991) Coronin, an actin binding protein of Dictyostelium discoideumlocalized to cell surface projections, has sequence similarities to Gprotein � subunits. EMBO J. 10, 4097–4104.

289. de Hostos, E. L., Rehfuess, C., Bradtke, B., Waddell, D. R., Albrecht, R.,Murphy, J., Gerisch, G. (1993) Dictyostelium mutants lacking the cy-toskeletal protein coronin are defective in cytokinesis and cell motility.J. Cell Biol. 120, 163–173.

290. Grogan, A., Reeves, E., Keep, N., Wientjes, F., Totty, N. F., Burlingame,A. L., Hsuan, J. J., Segal, A. W. (1997) Cytosolic phox proteins interactwith and regulate the assembly of coronin in neutrophils. J. Cell Sci.110, 3071–3081.

291. Maniak, M., Rauchenberger, R., Albrecht, R., Murphy, J., Gerisch, G.(1995) Coronin involved in phagocytosis: dynamics of particle-inducedrelocalization visualized by a green fluorescent protein Tag. Cell 83,915–924.

292. Gerisch, G., Albrecht, R., Heizer, C., Hodgkinson, S., Maniak, M. (1995)Chemoattractant-controlled accumulation of coronin at the leading edge


of Dictyostelium cells monitored using a green fluorescent protein-coronin fusion protein. Curr. Biol. 5, 1280–1285.

293. Wientjes, F. B., Reeves, E. P., Soskic, V., Furthmayr, H., Segal, A. W.(2001) The NADPH oxidase components p47(phox) and p40(phox) bindto moesin through their PX domain. Biochem. Biophys. Res. Commun.289, 382–388.

294. Suzuki, K., Yamaguchi, T., Oshizawa, T., Yamamoto, Y., Nishimaki-Mogami, T., Hayakawa, T., Takahashi, A. (1995) Okadaic acid inducesboth augmentation and inhibition of opsonized zymosan-stimulated su-peroxide production by differentiated HL-60 cells. Possible involvementof dephosphorylation of a cytosolic 21K protein in respiratory burst.Biochim. Biophys. Acta 1266, 261–267.

295. Djafarzadeh, S., Niggli, V. (1997) Signaling pathways involved in de-phosphorylation and localization of the actin-binding protein cofilin instimulated human neutrophils. Exp. Cell Res. 236, 427–435.

296. Heyworth, P. G., Robinson, J. M., Ding, J., Ellis, B. A., Badwey, J. A.(1997) Cofilin undergoes rapid dephosphorylation in stimulated neutro-phils and translocates to ruffled membranes enriched in products of theNADPH oxidase complex. Evidence for a novel cycle of phosphorylationand dephosphorylation. Histochem. Cell Biol. 108, 221–233.

297. Mukherjee, G., Quinn, M. T., Linner, J. G., Jesaitis, A. J. (1994)Remodeling of the plasma membrane after stimulation of neutrophilswith f-Met-Leu-Phe and dihydrocytochalasin B: identification of mem-brane subdomains containing NADPH oxidase activity. J. Leukoc. Biol.55, 685–694.

298. Touyz, R. M., Yao, G., Quinn, M. T., Pagano, P. J., Schiffrin, E. L. (2005)p47phox associates with the cytoskeleton through cortactin in humanvascular smooth muscle cells: role in NAD(P)H oxidase regulation byangiotensin II. Arterioscler. Thromb. Vasc. Biol. 25, 512–518.

299. Hartwig, J. H., Chambers, K. A., Stossel, T. P. (1989) Association ofgelsolin with actin filaments and cell membranes of macrophages andplatelets. J. Cell Biol. 108, 467–479.

300. Tamura, M., Kai, T., Tsunawaki, S., Lambeth, J. D., Kameda, K. (2000)Direct interaction of actin with p47(phox) of neutrophil NADPH oxidase.Biochem. Biophys. Res. Commun. 276, 1186–1190.

301. Hoeller, D., Volarevic, S., Dikic, I. (2005) Compartmentalization ofgrowth factor receptor signaling. Curr. Opin. Cell Biol. 17, 107–111.

302. Burack, W. R., Shaw, A. S. (2000) Signal transduction: hanging on ascaffold. Curr. Opin. Cell Biol. 12, 211–216.

303. Mukherjee, S., Maxfield, F. R. (2004) Membrane domains. Annu. Rev.Cell Dev. Biol. 20, 839–866.

304. Wei, Y., Yang, X., Liu, Q., Wilkins, J. A., Chapman, H. A. (1999) A rolefor caveolin and the urokinase receptor in integrin-mediated adhesionand signaling. J. Cell Biol. 144, 1285–1294.

305. Brown, D., Waneck, G. L. (1992) Glycosyl-phosphatidylinositol-an-chored membrane proteins. J. Am. Soc. Nephrol. 3, 895–906.

306. Rose, J. J., Foley, J. F., Murphy, P. M., Venkatesan, S. (2004) On themechanism and significance of ligand-induced internalization of humanneutrophil chemokine receptors CXCR1 and CXCR2. J. Biol. Chem.279, 24372–24386.

307. Hussain, N. K., Jenna, S., Glogauer, M., Quinn, C. C., Wasiak, S.,Guipponi, M., Antonarakis, S. E., Kay, B. K., Stossel, T. P., Lamarche-Vane, N., McPherson, P. S. (2001) Endocytic protein intersectin-l reg-ulates actin assembly via Cdc42 and N-WASP. Nat. Cell Biol. 3,927–932.

308. Barlic, J., Khandaker, M. H., Mahon, E., Andrews, J., DeVries, M. E.,Mitchell, G. B., Rahimpour, R., Tan, C. M., Ferguson, S. S., Kelvin, D. J.(1999) �-Arrestins regulate interleukin-8-induced CXCR1 internaliza-tion. J. Biol. Chem. 274, 16287–16294.

309. Fessler, M. B., Arndt, P. G., Frasch, S. C., Lieber, J. G., Johnson, C. A.,Murphy, R. C., Nick, J. A., Bratton, D. L., Malcolm, K. C., Worthen, G. S.(2004) Lipid rafts regulate lipopolysaccharide-induced activation ofCdc42 and inflammatory functions of the human neutrophil. J. Biol.Chem. 279, 39989–39998.

310. Shao, D., Segal, A. W., Dekker, L. V. (2003) Lipid rafts determineefficiency of NADPH oxidase activation in neutrophils. FEBS Lett. 550,101–106.

311. Barabe, F., Rollet-Labelle, E., Gilbert, C., Fernandes, M. J., Naccache,S. N., Naccache, P. H. (2002) Early events in the activation of Fc RIIAin human neutrophils: stimulated insolubilization, translocation to deter-gent-resistant domains, and degradation of Fc RIIA. J. Immunol. 168,4042–4049.

312. Katsumata, O., Hara-Yokoyama, M., Sautes-Fridman, C., Nagatsuka, Y.,Katada, T., Hirabayashi, Y., Shimizu, K., Fujita-Yoshigaki, J., Sugiya,H., Furuyama, S. (2001) Association of FcRII with low-density deter-gent-resistant membranes is important for cross-linking-dependent ini-tiation of the tyrosine phosphorylation pathway and superoxide genera-tion. J. Immunol. 167, 5814–5823.

313. Sengelov, H., Voldstedlund, M., Vinten, J., Borregaard, N. (1998) Humanneutrophils are devoid of the integral membrane protein caveolin. J. Leu-koc. Biol. 63, 563–566.

314. Newburger, P. E., Chovaniec, M. E., Greenberger, J. S., Cohen, H. J.(1979) Functional changes in human leukemic cell line HL-60. A modelfor myeloid differentiation. J. Cell Biol. 82, 315–322.

315. Harris, J., Werling, D., Koss, M., Monaghan, P., Taylor, G., Howard, C. J.(2002) Expression of caveolin by bovine lymphocytes and antigen-presenting cells. Immunology 105, 190–195.

316. Borregaard, N. (1988) Subcellular localization and dynamics of compo-nents of the respiratory burst oxidase. J. Bioenerg. Biomembr. 20,637–651.

317. Dahlgren, C., Follin, P. (1990) Degranulation in human neutrophilsprimes the cells for subsequent responsiveness to the chemoattractantN-formylmethionylleucylphenylalanine but does not increase the sensi-tivity of the NADPH-oxidase to an intracellular calcium rise. Biochim.Biophys. Acta 1052, 42–46.

318. Fossati, G., Bucknall, R. C., Edwards, S. W. (2002) Insoluble and solubleimmune complexes activate neutrophils by distinct activation mecha-nisms: changes in functional responses induced by priming with cyto-kines. Ann. Rheum. Dis. 61, 13–19.

319. Jones, H. A., Cadwallader, K. A., White, J. F., Uddin, M., Peters, A. M.,Chilvers, E. R. (2002) Dissociation between respiratory burst activity anddeoxyglucose uptake in human neutrophil granulocytes: implications forinterpretation of (18)F-FDG PET images. J. Nucl. Med. 43, 652–657.

320. Videm, V., Nilsson, L., Venge, P., Svennevig, J. L. (1991) Reducedgranulocyte activation with a heparin-coated device in an in vitro modelof cardiopulmonary bypass. Artif. Organs 15, 90–95.

321. Curnutte, J. T., Kuver, R., Scott, P. J. (1987) Activation of neutrophilNADPH oxidase in a cell-free system. Partial purification of componentsand characterization of the activation process. J. Biol. Chem. 262,5563–5569.

322. Sergeant, S., McPhail, L. C. (1997) Opsonized zymosan stimulates theredistribution of protein kinase C isoforms in human neutrophils. J. Im-munol. 159, 2877–2885.

323. Dri, P., Cramer, R., Menegazzi, R., Patriarca, P. (1985) Increased de-granulation of human myeloperoxidase-deficient polymorphonuclear leu-cocytes. Br. J. Haematol. 59, 115–125.

324. Humphreys, J. M., Davies, B., Hart, C. A., Edwards, S. W. (1989) Roleof myeloperoxidase in the killing of Staphylococcus aureus by humanneutrophils: studies with the myeloperoxidase inhibitor salicylhydrox-amic acid. J. Gen. Microbiol. 135, 1187–1193.

325. Levitz, S. M., Farrell, T. P. (1990) Human neutrophil degranulationstimulated by Aspergillus fumigatus. J. Leukoc. Biol. 47, 170–175.

326. Djaldetti, M., Salman, H., Bergman, M., Djaldetti, R., Bessler, H. (2002)Phagocytosis—the mighty weapon of the silent warriors. Microsc. Res.Tech. 57, 421–431.

327. Mansfield, P. J., Hinkovska-Galcheva, V., Carey, S. S., Shayman, J. A.,Boxer, L. A. (2002) Regulation of polymorphonuclear leukocyte degran-ulation and oxidant production by ceramide through inhibition of phos-pholipase D. Blood 99, 1434–1441.

328. Aiboshi, J., Moore, E. E., Ciesla, D. J., Silliman, C. C. (2001) Bloodtransfusion and the two-insult model of post-injury multiple organ failure.Shock 15, 302–306.

329. Regier, D. S., Waite, K. A., Wallin, R., McPhail, L. C. (1999) Aphosphatidic acid-activated protein kinase and conventional proteinkinase C isoforms phosphorylate p22(phox), an NADPH oxidase compo-nent. J. Biol. Chem. 274, 36601–36608.

330. Babior, B. M. (1978) Oxygen-dependent microbial killing by phagocytes(first of two parts). N. Engl. J. Med. 298, 659–668.

331. Lofgren, R., Serrander, L., Forsberg, M., Wilsson, A., Wasteson, A.,Stendahl, O. (1999) CR3, FcRIIA and FcRIIIB induce activation ofthe respiratory burst in human neutrophils: the role of intracellularCa(2�), phospholipase D and tyrosine phosphorylation. Biochim. Bio-phys. Acta 1452, 46–59.

332. DeChatelet, L. R., Shirley, P. S., Johnston Jr., R. B. (1976) Effect ofphorbol myristate acetate on the oxidative metabolism of human poly-morphonuclear leukocytes. Blood 47, 545–554.

333. Akard, L. P., English, D., Gabig, T. G. (1988) Rapid deactivation ofNADPH oxidase in neutrophils: continuous replacement by newly acti-vated enzyme sustains the respiratory burst. Blood 72, 322–327.

334. Leusen, J. H., Bolscher, B. G., Hilarius, P. M., Weening, R. S., Kaul-fersch, W., Seger, R. A., Roos, D., Verhoeven, A. J. (1994) 156Pro 3Gln substitution in the light chain of cytochrome b558 of the humanNADPH oxidase (p22-phox) leads to defective translocation of the cyto-solic proteins p47-phox and p67-phox. J. Exp. Med. 180, 2329–2334.

335. Massenet, C., Chenavas, S., Cohen-Addad, C., Dagher, M. C., Brandolin,G., Pebay-Peyroula, E., Fieschi, F. (2005) Effects of p47phox C terminus


phosphorylations on binding interactions with p40phox and p67phox:structural and functional comparison of p40phox and p67phox SH3domains. J. Biol. Chem. 280, 13752–13761.

336. Nisimoto, Y., Freeman, J. L., Motalebi, S. A., Hirshberg, M., Lambeth,J. D. (1997) Rac binding to p67(phox). Structural basis for interactions ofthe Rac1 effector region and insert region with components of therespiratory burst oxidase. J. Biol. Chem. 272, 18834–18841.

337. Quinn, M. T., Parkos, C. A., Jesaitis, A. J. (1995) Purification of humanneutrophil NADPH oxidase cytochrome b-558 and association with Rap1A. Methods Enzymol. 255, 476–487.

338. Maridonneau-Parini, I., Tringale, S. M., Tauber, A. I. (1986) Identifica-tion of distinct activation pathways of the human neutrophil NADPH-oxidase. J. Immunol. 137, 2925–2929.

339. Lockney, M. W., Golomb, H. M., Dawson, G. (1982) Unique cell surfaceglycoprotein expression in hairy cell leukemia: effect of phorbol estertumor promoters on surface glycoproteins, cell morphology, and adher-ence. Cancer Res. 42, 3724–3728.

340. Solanki, V., Rana, R. S., Slaga, T. J. (1981) Diminution of mouseepidermal superoxide dismutase and catalase activities by tumor pro-moters. Carcinogenesis 2, 1141–1146.

341. Varshavsky, A. (1981) Phorbol ester dramatically increases incidence ofmethotrexate-resistant mouse cells: possible mechanisms and relevanceto tumor promotion. Cell 25, 561–572.

342. Seifert, R., Schachtele, C. (1988) Studies with protein kinase C inhibitorspresently available cannot elucidate the role of protein kinase C in theactivation of NADPH oxidase. Biochem. Biophys. Res. Commun. 152,585–592.

343. Kim, J. S., Nam, J. S., Chae, H. D., Kim, K. T. (1997) A protein kinaseC-activating phorbol ester enhances transcription of the human DBHgene through a cyclic AMP response element in SK-N-BE(2)C cells.Brain Res. Mol. Brain Res. 51, 154–160.

344. Bokoch, G. M. (1995) Chemoattractant signaling and leukocyte activa-tion. Blood 86, 1649–1660.

345. Morel, F., Doussiere, J., Vignais, P. V. (1991) The superoxide-generatingoxidase of phagocytic cells. Physiological, molecular and pathologicalaspects. Eur. J. Biochem. 201, 523–546.

346. Cockcroft, S. (1992) G-protein-regulated phospholipases C, D and A2-mediated signaling in neutrophils. Biochim. Biophys. Acta 1113, 135–160.

347. Volpi, M., Yassin, R., Naccache, P. H., Sha’afi, R. I. (1983) Chemotacticfactor causes rapid decreases in phosphatidylinositol,4,5-bisphosphateand phosphatidylinositol 4-monophosphate in rabbit neutrophils. Bio-chem. Biophys. Res. Commun. 112, 957–964.

348. Takenawa, T., Ishitoya, J., Homma, Y., Kato, M., Nagai, Y. (1985) Roleof enhanced inositol phospholipid metabolism in neutrophil activation.Biochem. Pharmacol. 34, 1931–1935.

349. Smith, C. D., Lane, B. C., Kusaka, I., Verghese, M. W., Snyderman, R.(1985) Chemoattractant receptor-induced hydrolysis of phosphatidylino-sitol 4,5-bisphosphate in human polymorphonuclear leukocyte mem-branes. Requirement for a guanine nucleotide regulatory protein. J. Biol.Chem. 260, 5875–5878.

350. Ishitoya, J., Yamakawa, A., Takenawa, T. (1987) Translocation of diacyl-glycerol kinase in response to chemotactic peptide and phorbol ester inneutrophils. Biochem. Biophys. Res. Commun. 144, 1025–1030.

351. Rossi, F., Grzeskowiak, M., Della Bianca, V., Calzetti, F., Gandini, G.(1990) Phosphatidic acid and not diacylglycerol generated by phospho-lipase D is functionally linked to the activation of the NADPH oxidase byfMLP in human neutrophils. Biochem. Biophys. Res. Commun. 168,320–327.

352. Pai, J. K., Siegel, M. I., Egan, R. W., Billah, M. M. (1988) PhospholipaseD catalyzes phospholipid metabolism in chemotactic peptide-stimulatedHL-60 granulocytes. J. Biol. Chem. 263, 12472–12477.

353. Agwu, D. E., McPhail, L. C., Chabot, M. C., Daniel, L. W., Wykle, R. L.,McCall, C. E. (1989) Choline-linked phosphoglycerides. A source ofphosphatidic acid and diglycerides in stimulated neutrophils. J. Biol.Chem. 264, 1405–1413.

354. Gelas, P., Ribbes, G., Record, M., Terce, F., Chap, H. (1989) Differentialactivation by fMet-Leu-Phe and phorbol ester of a plasma membranephosphatidylcholine-specific phospholipase D in human neutrophil.FEBS Lett. 251, 213–218.

355. Djerdjouri, B., Lenoir, M., Giroud, J. P., Perianin, A. (1999) Contributionof mitogen-activated protein kinase to stimulation of phospholipase D bythe chemotactic peptide fMet-Leu-Phe in human neutrophils. Biochem.Biophys. Res. Commun. 264, 371–375.

356. Erickson, R. W., Langel-Peveri, P., Traynor-Kaplan, A. E., Heyworth,P. G., Curnutte, J. T. (1999) Activation of human neutrophil NADPHoxidase by phosphatidic acid or diacylglycerol in a cell-free system.Activity of diacylglycerol is dependent on its conversion to phosphatidicacid. J. Biol. Chem. 274, 22243–22250.

357. Bromberg, Y., Pick, E. (1983) Unsaturated fatty acids as second mes-sengers of superoxide generation by macrophages. Cell. Immunol. 79,240–252.

358. Maridonneau-Parini, I., Tauber, A. I. (1986) Activation of NADPH-oxidase by arachidonic acid involves phospholipase A2 in intact humanneutrophils but not in the cell-free system. Biochem. Biophys. Res.Commun. 138, 1099–1105.

359. Dana, R., Malech, H. L., Levy, R. (1994) The requirement for phospho-lipase A2 for activation of the assembled NADPH oxidase in humanneutrophils. Biochem. J. 297, 217–223.

360. Henderson, L. M., Moule, S. K., Chappell, J. B. (1993) The immediateactivator of the NADPH oxidase is arachidonate not phosphorylation.Eur. J. Biochem. 211, 157–162.

361. Lowenthal, A., Levy, R. (1999) Essential requirement of cytosolic phos-pholipase A(2) for activation of the H(�) channel in phagocyte-like cells.J. Biol. Chem. 274, 21603–21608.

362. Hazan-Halevy, I., Seger, R., Levy, R. (2000) The requirement of bothextracellular regulated kinase and p38 mitogen-activated protein kinasefor stimulation of cytosolic phospholipase A(2) activity by eitherFcRIIA or FcRIIIB in human neutrophils. A possible role for Pyk2 butnot for the Grb2-Sos-Shc complex. J. Biol. Chem. 275, 12416–12423.

363. Yagawa, K., Kaku, M., Ichinose, Y., Aida, Y., Tomoda, A. (1985) Fcreceptor-mediated desensitization of superoxide (O2–) generation re-sponse of guinea-pig macrophages and polymorphonuclear leucocytes.Immunology 55, 629–638.

364. Worku, M., Paape, M. J., Di, C. A., Kehrli Jr., M. E., Marquardt, W. W.(1995) Complement component C3b and immunoglobulin Fc receptorson neutrophils from calves with leukocyte adhesion deficiency. Am. J.Vet. Res. 56, 435–439.

365. Botha, A. J., Moore, F. A., Moore, E. E., Kim, F. J., Banerjee, A.,Peterson, V. M. (1995) Postinjury neutrophil priming and activation: anearly vulnerable window. Surgery 118, 358–364.

366. Botha, A. J., Moore, F. A., Moore, E. E., Sauaia, A., Banerjee, A.,Peterson, V. M. (1995) Early neutrophil sequestration after injury: apathogenic mechanism for multiple organ failure. J. Trauma 39, 411–417.

367. Ousman, S. S., David, S. (2000) Lysophosphatidylcholine induces rapidrecruitment and activation of macrophages in the adult mouse spinalcord. Glia 30, 92–104.


Structural organization of the neutrophil NADPH oxidase ...

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