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Oxidative killing of microbes by neutrophils
Microbes and Infection 5 (2003) 1307–1315 www.elsevier.com/locate/micinf
Forum in immunology
Oxidative killing of microbes by neutrophils Dirk Roos a,*, Robin van Bruggen a,b, Christof Meischl a,1 a
Sanquin Research at CLB, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands b Emma Children’s Hospital, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
Abstract Neutrophils and other phagocytic leukocytes contain a phagocyte NADPH oxidase enzyme that generates superoxide after cell activation. Reactive oxygen species derived from superoxide, together with proteases liberated from the granules, are used to kill ingested microbes. Dysfunction of the phagocyte NADPH oxidase results in chronic granulomatous disease, with life-threatening infections. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Neutrophils; Microbes; Killing; NADPH oxidase; Chronic granulomatous disease
1. Introduction Neutrophils fulfill their role as effector cells mainly by internalizing (phagocytosing) pathogens and exposing them to two destructive principles: reactive oxygen species (ROS) generated by an enzyme called the phagocyte NADPH oxidase 2 and hydrolytic granule proteins. The importance of these systems is exemplified by the clinical symptoms displayed by chronic granulomatous disease (CGD) patients with a dysfunctional NADPH oxidase in their phagocytes [1] and by mouse models in which elastase and cathepsin G have been knocked out [2]. Given their potential for destruction, it is not surprising that neutrophils and the organism as a whole have developed tight control and safety mechanisms to minimize the risk of neutrophil-inflicted damage to the organism. Examples of these control and safety mechanisms are the tight regulation of the activation of the NADPH oxidase and the high concentrations of proteinase inhibitors in plasma and tissue fluids. However, the protection thus obtained is not absolute, and well-known examples of disease states accom* Corresponding author. Tel.: +31-20-51233377; fax: +31-20-5123474. E-mail address: [email protected] (D. Roos). 1 Present address: Department of Pathology, Free University Medical Centre, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. 2 The NADPH oxidase enzyme present in phagocytic leukocytes (neutrophils, eosinophils, monocytes/macrophages) and in Epstein–Barr virus (EBV)-transformed B lymphocytes, which is dysfunctional in patients with chronic granulomatous disease, is called the “phagocyte NADPH oxidase” in this review, to distinguish it from similar, homologous systems in other tissues (see Section 7). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2003.09.009
panied by tissue damage due to neutrophil-derived products include gout, chronic obstructive pulmonary disease, rheumatoid arthritis, autoimmune vasculitis and some forms of glomerulonephritis. 2. The phagocyte NADPH oxidase The phagocyte NADPH oxidase is a multicomponent enzyme with a redox center that transfers electrons from cytoplasmic NADPH onto extracellular (or intraphagosomal) molecular oxygen, thereby generating superoxide (Fig. 1). The overall reaction catalyzed by this enzyme is: NADPH + 2O2 → NADP+ + 2O2– + H+ The electron transfer from NADPH to oxygen is a multistep process, during which the electrons are transported sequentially along several moieties of the oxidase: NADPH → FAD → 2 Heme → 2O2 Although FAD and the two heme groups are part of the redox center of the enzyme, cytochrome b558, NADPH cannot bind to this protein unless the complete enzyme has been assembled during activation, and only then can electron transfer actually take place. Cytochrome b558, a flavo-hemeprotein, is composed of two subunits, gp91phox and p22phox, in a 1:1 stoichiometry. Incorporated in the membranes of specific granules and secretory vesicles in resting cells, cytochrome b558 becomes expressed on the membrane of the phagolysosome and on the cell surface when the granules/vesicles fuse with those larger
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Fig. 1. Schematic illustration of the electron transfer mechanism of the phagocyte NADPH oxidase. After assembly of the NADPH oxidase complex, NADPH from the cytosol can bind to the enzyme and donate its electrons. These electrons are then transmitted via FAD and two heme groups to molecular oxygen on the outside of the plasma membrane, thus generating superoxide in either the phagosome or in the extracellular environment. Reproduced with permission by Springer-Verlag Heidelberg from Ref. [31].
membrane systems during cell activation (Fig. 2). The stimulus for this activation is the binding of opsonized microorganisms or high concentrations of chemoattractants to phagocyte surface receptors. As part of this activation, the enzyme’s three cytosolic components, p47phox, p67phox and p40phox, as well as a low-molecular-weight GTP-binding protein, translocate to the cytochrome b558 in the membrane to form there the complete and active form of the NADPH oxidase (Fig. 2). A defect in any one of the four components, gp91phox, p22phox, p47phox or p67phox, abolishes (or reduces) the activity of the oxidase and leads to CGD. Defects in the enzyme component p40phox are not known. Recently, a human immunodeficiency syndrome with impaired neutrophil chemotaxis, polarization, azurophilic granule secretion and superoxide production has been shown to be caused by a dominantly negative mutation in RAC2 [3].
2.1. Phagocyte NADPH oxidase components 2.1.1. gp91phox The CYBB gene that codes for gp91phox is located on the X chromosome (Xp21.1), has a length of 30 kb and comprises 13 exons. The translation product, a protein of 570 amino acids, needs for its further maturation and stabilization the presence of p22phox. Thus, abolished expression of one protein automatically leads to simultaneous absence of the other. Posttranslational modifications are the glycosylation of three of its five potential N-linked glycosylation sites. The N-terminal half of the mature protein contains six hydrophobic, probably membrane-spanning domains, while the hydrophilic C-terminal part is thought to represent the cytosolic side of the protein. The hydrophobic part contains the heme moieties and is probably also involved in the interaction with
Fig. 2. Activation of the phagocyte NADPH oxidase. Assembly of the enzyme and phagosome formation are concomitant processes. Translocation of the cytosolic oxidase components is initiated by serine phosphorylation in p47phox and controlled by small Rho-like GTPases (Rac1, Rac2, Rap1A). This translocation leads to a conformational change in gp91phox that permits NADPH binding, thus activating the NADPH oxidase enzyme. Reproduced with permission by Springer-Verlag Heidelberg from Ref. [31].
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p22phox. For the C-terminal part, a three-dimensional model has been deduced from sequence homology with the ferridoxin NADP+ reductase flavoenzyme family. This part of the protein contains one binding site for NADPH and the FADbinding site. In the inactive state of the enzyme, the NADPHbinding site is probably covered by a loop of 20 amino acids. 2.1.2. p22phox p22phox is encoded by the gene CYBA, which is located on chromosome 16q24 and spans 8.5 kb and six exons. The resulting protein of 195 amino acids has one proline-rich region involved in interaction with SH3 (src homology region 3) domains. 2.1.3. p47phox NCF1, the gene on chromosome 7q11.23 coding for p47phox, has a length of 15 kb, encompassing 11 exons. Its product, a protein of 390 amino acids, contains nine serine phosphorylation sites, two SH3 domains, one PX (Phox homology) domain involved in targeting membranes by binding to phosphoinositides, and one proline-rich region. 2.1.4. p67phox NCF2, on chromosome 1q25 codes for p67phox, is 40 kb long and comprises 16 exons. p67phox itself, with its 526 amino acids, possesses a (higher affinity) binding site for NADPH, four tetratricopeptide repeat (TPR) motifs involved in binding of Rac1 or Rac2, two SH3 domains and one proline-rich region. 2.1.5. p40phox Very little is known yet about p40phox, a protein of 339 amino acids. Its gene, NCF4, spans 18 kb and 10 exons and is located on chromosome 22q13.1. The protein contains one SH3 and one PX domain. It probably plays a role both in stabilization of the p47phox/p67phox complex in the cytosol and in facilitating membrane recruitment of this complex during NADPH oxidase activation. 2.2. Activation of the enzyme Responding to as yet only partially unravelled upstream events that transmit the signals originating from the cell surface receptors, the activation of the NADPH oxidase itself seems to be initiated by a change in the phosphorylation of p47phox. The different protein kinases implicated in this process apparently phosphorylate different groups of serines, with each different state of phosphorylation corresponding to a different three-dimensional conformation of the protein. This process disrupts the cytosolic complex of p47phox/ p67phox/p40phox in the resting cell, exposing until then inaccessible SH3 and proline-rich domains, as well as PX domains. This change results in translocation of these three proteins to the membrane, where they associate with cytochrome b558 (Fig. 2). The interactions of the cytosolic components amongst each other and with the cytochrome are mediated by SH3 domains binding specifically to certain proline-rich
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regions, while the interactions of the cytosolic oxidase components with the membrane are PX-domain mediated. In the membrane-associated complex, p47phox appears to stabilize the interaction of p67phox with the cytochrome. p67phox with its high-affinity binding site for NADPH, on the other hand, could bind to gp91phox, which contains a lower affinity NADPH-binding site, to form the catalytically efficient binding site of the active enzyme. The loop of 20 amino acids that covers the NADPH-binding site in gp91phox in the resting state is thought to move away, as a result either of the complex formation or of an independent control mechanism. NADPH then binds to the completely assembled oxidase, electron transfer will start and superoxide generation begins (Fig. 1). Both the assembly of the enzyme and the electron flow itself are subject to regulating and modifying influences of three low-molecular-weight GTP-binding proteins, Rac1, Rac2 and Rap1A [4,5]. Functioning as molecular switches in signaling cascades, with an inactive GDP-bound and an active GTPbound state, these Rho-like GTPases themselves are again under the control of guanine-nucleotide exchange factors (GEFs) and GTPase-activating (GAP) proteins. In neutrophils, one Rac-activating GEF has been identified: P-Rex1, which is synergistically activated by phosphoinositides and bc subunits of trimeric G proteins associated with seven-span membrane receptors [6]. This indicates that P-Rex1 is under direct control of ligand (e.g. chemokine) binding to these receptors. Upon activation of Rac, these GTPases are liberated from Rho-GDI (GDP dissociation inhibitor), and as a result, a conserved carboxyl-terminal 15-carbon farnesyl group becomes exposed that mediates association of Rac with the membrane and thus with the cytochrome. Rac1 (expressed predominantly in macrophages) and Rac2 (expressed predominantly in neutrophils) interact with two of the TPR domains of p67phox. In the activated oxidase complex, Rac participates in and modulates the electron transfer steps from NADPH to FAD, heme and onto molecular oxygen. GTPase-activating proteins, finally, seem to play a role in the deactivation process of the NADPH oxidase. Few data are as yet available about the role of Rap1A in the activation of the oxidase. Rap1A associates with the NADPH oxidase in a phosphorylation-dependent manner, and clearly is involved in the regulation process, as both constitutively active or inactive mutants decrease or inhibit the respiratory burst, and overexpression of the wild-type GTP-binding protein enhances O2– production [7]. Rap1A itself is activated after stimulation of neutrophils with a variety of stimuli, including fMet–Leu–Phe (fMLP), platelet-activating factor (PAF), granulocyte-macrophage colony-stimulating factor (GMCSF), and IgG-coated particles, involving both phospholipase C-dependent and -independent pathways. Furthermore, Rap1A seems to activate protein kinase C and may, therefore, also have a more indirect role in oxidase regulation. Although it is becoming increasingly clear that interactions with other proteins—possibly in the form of a macromolecular complex—and with the cytoskeleton [8] must play an important role in the regulation and the localization of the
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oxidase activity, almost nothing is known yet about this cellular background of NADPH oxidase regulation. In addition, arachidonic acid, liberated by cytosolic phospholipase A2 from plasma membrane phopholipids, seems to play an essential role in oxidase activation [9]. Whether arachidonic acid itself exerts this effect on one or more of the oxidase components or whether the membrane lysophospholipids create a microenvironment suitable for oxidase complex formation is unknown. The impressive complexity of the activation of the enzyme and its intricate control mechanisms point to the importance of a tightly controlled, place- and time-restricted release of free oxygen radicals, since an uncontrolled release of these products would have devastating effects for the affected individual. This complexity, on the other hand, is the explanation for the fact that defects in different genes can lead to the same cellular dysfunction and disease. 3. Chronic granulomatous disease 3.1. Clinical presentation CGD is a rare disease, with an estimated incidence of 1:250,000, all ethnic groups being equally affected. The most common form of the syndrome is X-linked, and the overwhelming majority of these patients is, obviously, male; in the autosomal forms, no sex preference is discernible. CGD presents early in life in the form of acute or chronic infections, two-thirds of the patients showing the first symptoms already in their first year of life [1]. As a general rule, the organs that represent the individual’s border against the outside world, or the lymph stations downstream of those organs, are most frequently affected: lungs, skin and gastrointestinal tract. The isolated pathogens are usually catalase positive and/or relatively resistant to the other, non-oxidative killing mechanisms of the phagocytes. Catalase degrades hydrogen peroxide (H2O2), which is also produced in small amounts by the microbes themselves, and thus deprives the phagocytes of the possibility to use this microbe-generated oxygen metabolite for killing. The microorganisms most frequently found in CGD patients are Staphylococcus aureus, various Aspergillus species, enteric Gram-negative bacteria (including Serratia marcescens and various Salmonella species) and Burkholderia cepacia. Frequently encountered infections are (in descending order of frequency) pneumonia, cutaneous abscesses and suppurative lymphadenitis, hepatic and perihepatic abscesses and osteomyelitis. While the above affections normally represent acute disease states, CGD—as the name implies—is typically characterized by a chronic struggle of the immune system with the pathogens. The granulomas that can be found in a large variety of organs and to which CGD owes its name are the result of chronic inflammatory cell reactions, involving mainly lymphocytes and histiocytes. Clinically, the granulomas can become symptomatic by pain or signs of obstruction of the gastrointestinal or urinary tract. Other manifestations
of chronic inflammation are the inflammatory bowel disease of CGD, which closely resembles Crohn’s disease, an eczematoid dermatitis and, very rarely, discoid or, even more rarely, systemic lupus erythematosus. 3.2. Molecular defects in CGD 3.2.1. Defects in gp91phox All possible types of mutation, except gene conversions, have been found in CYBB, with single-nucleotide substitutions accounting for 65% of the defects, and deletions and insertions for the remaining 35%. Very large deletions, extending over other coding genes localized on the X chromosome, can result in various clinical entities, such as Duchenne muscular dystrophy, retinitis pigmentosa or McLeod’s syndrome, being associated with CGD. In a multicenter review of the mutations found in 261 X-linked CGD kindreds, 65% of these mutations were found to be family specific, with the other 35% being clustered around a few hotspots, mainly around CpG sequences [1]. The large majority of X-linked mutations in CGD lead to a complete lack of gp91phox, due to instability of the mRNA or of the translated protein. These are called X910 variants, to differentiate them from the (few) cases with reduced or normal protein expression, called X91– and X91+, respectively. In the cases of X91–, the reduced protein expression is accompanied by a roughly proportional decrease in superoxide production, whereas the X91+ variants express normal amounts of a non-functional protein. While being clinically indistinguishable from the X910 variants, the cases of X91+ CGD are of great interest for understanding the working mechanism of the oxidase, because they allow analysis of how different defects block various steps in the activation process or in the electron transport. The 15 X91+ mutations known so far [10] have contributed in this way to our knowledge of gp91phox, by identifying regions important for the binding of one of the heme groups, NADPH or the cytosolic oxidase components. Five families have been found with single nucleotide substitutions in the 5′ promoter region of CYBB. These mutations are clustered in the region around bp-55, and each ablates a binding site recognized by the Ets transcription factors PU.1 and Elf-1 [11]. In three of these families, with mutations at –52 or –53, the eosinophils still expressed gp91phox and oxidase activity, whereas the neutrophils, monocytes and EBV-transformed B lymphocytes were defective; this has led to the characterization of eosinophilspecific regulation of gp91phox gene expression by the transcription factors GATA-1 and GATA-2 [12]. 3.2.2. Defects in p22phox About 5% of the cases of CGD are caused by defects in p22phox. In the 34 families with A22-CGD investigated so far, 27 different mutations were found in the 68 alleles involved [13]. The only A22+-mutation known, a substitution of glutamic acid for one of the prolines in its proline-rich region, apparently destroys the interaction with the SH3
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domain of p47phox, thereby interrupting the activation of the enzyme. 3.2.3. Defects in p47phox Mutations in NCF1 account for about 30% of the cases of CGD. In strong contrast with the variation in the mutations found in the other subtypes of CGD, only a few different mutations have been reported in A47 CGD to date. In 52 unrelated patients described, 41 were homozygotes and eight compound heterozygotes for a dinucleotide deletion in the first four nucleotides of exon 2 (GTGT → GT) [13]; overall, 11 different mutations have been found in this patient group. This situation has long remained unexplained, but the finding that most wild-type alleles contain two pseudogenes—highly homologous but non-functional gene copies of p47phox—in addition to NCF1, has led to the realization that recombination events between NCF1 and a pseudogene cause the extremely high uniformity of mutations found in A47 CGD. 3.2.4. Defects in p67phox With around 5% of the described cases, A67 CGD also represents a rare subtype of the disease. In the 24 A67 CGD patients characterized thus far, 23 different mutations were found among the 48 alleles [13]. While the level of mRNA is usually normal, no protein expression has been found in A67 CGD, with the exception of one A67+ patient, whose p67phox protein is apparently non-functional due to the deletion of one amino acid. This deletion causes a strongly diminished binding of Rac, and thereby a disturbance in NADPH oxidase activation [14]. 3.2.5. Defects in p40phox There are no known defects in this protein. 3.3. Correlations between genotype and phenotype in CGD In general, the X910 and X91+ subforms of CGD present with similar clinical severity. It might be expected, on the other hand, that the severity of the clinical symptoms in X91– CGD correlates with the amount of residual superoxide production (3–30%) found in these patients’ phagocytes. This is, while often true, not a dependable rule. Probably, variabilities in other host-defense systems play an important role here, as borne out by a study that examined the role of host-defense molecule polymorphisms in determining immune-mediated complications in CGD. This study demonstrated, for example, strong associations of myeloperoxidase and mannosebinding lectin genotypes with gastrointestinal complications and autoimmune/rheumatologic disorders, respectively [15]. Other such associations quite certainly exist and determine cumulatively the overall phenotype. Autosomal CGD, as borne out by a few clinical studies, seems to follow in general a more benign clinical course [1]. Given the observations that p47phox, under certain in vitro conditions, seems not to be essential for oxidase activity, it might be speculated that some residual H2O2 generation is also possible in the in vivo situation.
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4. Function of the phagocyte NADPH oxidase It is clear that the defect in killing of microbes by neutrophils from patients with CGD results from the inability of these cells to generate superoxide. However, superoxide has a low bactericidal potency, so it is probably not superoxide itself that kills the microbes. Within the phagosome, superoxide is—spontaneously or enzymatically—converted into H2O2, which may then react with superoxide to generate hydroxyl radicals and singlet oxygen, both highly reactive and toxic compounds. Superoxide can also react with nitrogen oxide (NO), generated by inducible NO synthase, to yield peroxynitrite, a very reactive nitrogen intermediate. There are even indications that singlet oxygen may be converted into a compound similar to ozone (O3) in a reaction catalyzed by antibodies bound to microbes or neutrophils. H2O2 may also, together with chloride, be used as a substrate by myeloperoxidase released from azurophil granules to generate hypochlorous acid (also known as bleach), a very toxic compound for almost all microbes (Fig. 3). Subsequently, the short-lived hypochlorous acid can react with secondary amines to form secondary chloramines, which are as microbicidal as hypochlorous acid but much more stable. Is this then the way in which neutrophils kill so very efficiently the large array of microorganisms that we encounter? There is at present a debate in the literature whether this is really the case. Doubts about this concept have arisen from a number of observations. One is the fact that deficiency of myeloperoxidase is a common event but seldom leads to serious defects in the killing of microbes. Another is the observation that double knock-out mice for elastase and cathepsin G have a defect in bacterial killing similar to that of mice with a defect in the phagocyte NADPH oxidase [2]. This latter observation brought Tony Segal in London (UK) to the idea that the oxidase might be involved in the liberation of lysosomal proteases in the phagosome. Some 20 years ago already, Segal noticed that the pH in the phagosome initially increases to a value around pH 8 shortly after phagosome formation, despite the fusion of the phagosome with the acidic azurophil granules [16]. Only after about 30 min, does the intraphagosomal pH drop to values below 7. In contrast, in CGD neutrophils, the pH rapidly drops to values around 6.5. In the years to follow, it became clear that the leukocyte NADPH oxidase not only transfers electrons from NADPH in the cytosol to oxygen in the phagosome but also transfers protons from the cytosol to the phagosome, to compensate for this charge separation [17]. However, if this proton influx into the phagosome were equal to the electron influx, the pH would remain neutral, or decrease, due to fusion of the granules with the phagosome (Fig. 3). In fact, the pH increases, indicating that the proton influx is insufficient to compensate for the electron influx. Recently, Segal’s group published evidence that part of the charge compensation is due to influx of potassium ions instead of protons [2]. These investigators also found that these potassium ions are instrumental in liberating proteases such as elastase and cathepsin
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Fig. 3. Within the phagosome. In the small space between an ingested bacterium (shaded area) and the membrane of the phagosome, a number of chemical reactions take place. Molecular oxygen (O2) is reduced to superoxide (O2–) by electrons pumped into the phagosome by the phagocyte NADPH oxidase. This charge transfer is compensated by an influx of protons (H+) or other cations. The protons are used to reduce superoxide to H2O2, which can be degraded to oxygen and water in a catalase-dependent reaction. Alternatively, H2O2 can combine with chloride (Cl–) to form hypochlorous acid (HOCl) in a reaction catalyzed by myeloperoxidase (MPO). If all electrons pumped into the phagosome are compensated by proton influx, the pH in the phagosome will remain neutral. However, the pH does in fact rise to about pH 8, despite the release of acid contents from granules in the cytoplasm that fuse with the phagosome [16]. This indicates that other cations, such as potassium ions (K+), may enter the phagosome instead of protons [2]. If there is an influx of K+, these cations then mediate solubilization of proteases that are bound to the proteoglycan matrix of the granules. Reproduced with permission from Ref. [18].
G from their acidic proteoglycan matrix in the granules. Thus, these observations explain the increase in intraphagosomal pH to values optimal for protease action and indicate that the NADPH oxidase, rather than killing microorganisms by its oxidative products, acts by liberating lysosomal proteases. The debate now centers around the question whether the oxidative products do have a role for themselves in the microbial killing process or not [18]. A strong argument in favor of this concept is the observation that CGD neutrophils show enhanced killing of microorganisms when coingesting latex particles coated with glucose oxidase (which generates H2O2). In addition, bacteria in which catalase or superoxide dismutase have been inactivated are killed much more easily by neutrophils or by a cell-free, O2–- or H2O2-generating system than wild-type bacteria. Moreover, the killing of bacteria by H2O2 in a cell-free system is dramatically enhanced by myeloperoxidase, and myeloperoxidase-deficient mice succumb to bacterial challenge. All this evidence points to a direct contribution of ROS to the killing process. Release of proteases from the granules can also take place in the absence of oxidase activity or even at low potassium concentrations, as indicated by the release of these enzymes into the extracellular environment and by the killing of some bacterial strains by neutrophils under anaerobic conditions. Most likely, therefore, the leukocyte NADPH oxidase induces microbial killing both in a direct way (via oxidative products) and in an indirect way (via liberation of proteases).
5. Escape of microorganisms from oxidative killing Microorganisms have developed several mechanisms to escape from the action of the phagocyte NADPH oxidase. One of these is a strong defense against the toxic oxygen metabolites, such as a high expression and even excretion of superoxide dismutase, increased expression of catalase or other, as yet unidentified defense systems. For instance, Mycobacterium tuberculosis contains several noxR genes that confer resistance against both H2O2 and reactive nitrogen intermediates when expressed in heterologous bacteria [19]. The mode of action of the proteins encoded by these genes is unknown. Recently, we have identified a Salmonella typhimurium variant with decreased resistance against H2O2 due to the inactivation of Sspj, a pyrroloquinoline quinone (PQQ)-containing protein. Probably, this protein is involved in redox reactions leading to breakdown of H2O2. Salmonella species induce their own internalization into host cells by means of a set of proteins encoded by the salmonella pathogenicity island 1 (Spi-1). These proteins form a type-III secretion system for the introduction of bacterial proteins into the cytosol of the target cell. Spi-1 also harbors genes that encode proteins that are secreted into the target cell to modulate its actin cytoskeleton, leading to the uptake of the bacteria into a membrane-bound vacuole within the target cell. Within this salmonella-containing vacuole (SCV), the bacteria are able to resist the antimicrobial response of the infected host cells, among which are neutro-
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phils. This resistance is mediated by a second group of salmonella proteins, encoded by the Spi-2 gene cluster. These proteins interfere with the activation of the NADPH oxidase by preventing the formation of an enzymatically active oxidase complex [20,21]. Our own investigations have indicated that this pathogenicity system is not complete: although wild-type Salmonella prevents oxidase activation to a large extent, a limited amount of H2O2 is still generated within the vacuoles, and this amount of H2O2 is sufficient to damage the bacteria unless they are, in addition, protected by the PQQ-containing protein Sspj (and by superoxide dismutase and catalase). In addition, we found that opsonization of Salmonella with antibodies induces a 20–50-fold higher oxidase activity than that observed with unopsonized bacteria. This shows the importance of the mode of entrance of these bacteria in the activation of the NADPH oxidase. Another way of evading oxidative killing is shown by Anaplasma phagocytophila, an obligatory intracellular bacterium with a tropism for neutrophils, transmitted by ticks and causing human granulocytic ehrlichiosis. This microbe strongly inhibits the transcription of gp91phox and Rac2, leading to the loss of expression of these proteins and the inability of the infected cells to generate superoxide, even after incubation with phorbol esters [22]. This inhibition is essential for growth and survival of the bacteria within the neutrophils. However, during initial infection of neutrophils, A. phagocytophila must also be able to circumvent the oxidative attack. Indeed, superoxide release is inhibited for more than 90% already within 30 min of incubation of human neutrophils with these bacteria. The mechanism of this last process is unknown. 6. A possible role of the phagocyte NADPH oxidase in other tissues Numerous reports have been published that describe the presence of components of the phagocyte NADPH oxidase in cell types other than leukocytes, both in humans and in animals. Most notably, in cells of the vascular system (endothelial cells, vascular smooth muscle cells and fibroblasts), these components have been detected with various techniques, ranging from Northern blotting, RT-PCR and cDNA sequencing to immunohistochemistry and Western blotting. Interest in this subject stems from observations that ROS are involved in various (patho)physiological responses of vascular cells, such as mitosis, apoptosis, migration, hypertrophy and modifications of the extracellular matrix. Indeed, ROS have been implicated in several major intracellular signal transduction pathways leading to changes in gene transcription and protein synthesis. Therefore, ROS are supposed to be involved in disease processes such as atherosclerosis and tumor progression. Table 1 summarizes the evidence for the presence of gp91phox, p22phox, p47phox and p67phox in vascular cells of animal and human sources [23]. However, many of these studies suffer from the lack of appropriate negative controls, such as material from relevant knock-out mice or from CGD patients. In particular, the possibility of cross-
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reactivity of oligonucleotides or antibodies against these components with one or more of the NADPH oxidase component homologs (see next paragraph) has to be taken into account. So far, to our knowledge, no extra-phagocyte deficiencies of NADPH oxidase components have been detected in patients with CGD. For instance, CGD fibroblasts contain a spectroscopically normal cytochrome b558 content compared with control fibroblasts and generate comparable amounts of superoxide [24]. 7. Non-phagocytic NADPH oxidases Recently, several homologs of gp91phox have been identified in cells other than phagocytes. These homologs have conserved the overall structure of six transmembrane domains with four heme-coordinating histidines in the N-terminal part of the molecule, followed by a cytosolic C-terminus that contains highly conserved binding sites for FAD and NADPH. In a new nomenclature, these proteins are now called Nox (NADPH oxidase) proteins, including gp91phox (Nox2). Mox-1 (or Nox1) mRNA is expressed in colon, prostate, uterus and vascular smooth muscle cells [25]. ROS produced by this enzyme seem to be generated intracellularly, in contrast to the situation with the phagocyte NADPH oxidase. Overexpression of Nox1 results in increased cell growth, suggesting a role for these oxygen species in signal transduction for cell growth regulation. A role of this enzyme in intracellular defense against microorganisms cannot be excluded at present. Another gp91phox homolog, called Renox or Nox4, has been detected by in situ RNA hybridization and immunohistochemistry in the renal cortex, predominantly in proximal convoluted tubule epithelial cells [26]. It has been suggested that Nox4 may participate in the oxygen-sensing mechanism that regulates the production of erythropoietin. Transfection of Nox4 into 3T3 fibroblasts resulted in enhanced superoxide production; however, these modified cells displayed substantially diminished cell growth. Some of these alternative NADPH oxidases, specifically Nox1, may interact with specific cytosolic partners for activity regulation and localization, i.e., with the recently discovered alternative members of the p47 and p67 protein families [27,28]. Finally, two similar homologs have been discovered that are characterized by an N-terminal extension containing a peroxidase homology domain, a calmodulin-like calcium-binding motif and an additional transmembrane domain [29]. RNA for these proteins, called ThOX-1 and ThOX-2 (or Duox1 and Duox2), is expressed specifically in the thyroid gland, at the apical membrane of the thyrocytes. Duox2, and perhaps also Duox1, is involved in the iodination of the thyroid hormone, because nonsense mutations in the gene for Duox2 have been identified in patients with congenital hypothyroidism. Do phagocytes also contain alternative NADPH oxidases? There is one indication that they do indeed. Blouin et al. [30] have found that the signaling pathway for b2-integrin activation involves a redox reaction, because exogenous H2O2
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Table 1 NADPH oxidase components detected by various techniques Subunit Cell type (a) In vascular cells from animals ec p22phox vsmc
Western blot
Northern blot
RT-PCR
IHC
DNA sequencing
Anti-sense
r+
r+ r+
r, p + r+
r, p + r+ r– r, rb +
r+ r+
r+
r, p +
r, p +
r+
fb gp91phox
ec
r+
Spectroscopy
r–
p47phox
p67phox
vsmc fb
c+
ec vsmc fb
b+
r, rb + p, b + r, rb +
ec vsmc fb
p+ rb +
rb +
r, rb +
(b) In human vascular cells huvec p22phox
+
+
+
ca ec vsmc fb
+ + +
+
+
huvec
+
+
vsmc fb
–
–
p47phox
huvec vsmc fb
+ +
+ +
+
p67phox
huvec vsmc fb
+ –
+ –
+
gp91phox
+ + + +
–
+
–
Table reproduced with permission by the BMJ Publishing Group from Ref. [23]. Cell type: ec, endothelial cell; fb, fibroblast; vsmc, vascular smooth muscle cell; ca ec, coronary artery endothelial cell; huvec, human umbilical vein endothelial cell. Animal species: b, bovine; c, calf; m, mouse; p, porcine; r, rat; rb, rabbit. Techniques: IHC, immunohistochemistry; RT-PCR, reverse transcriptase polymerase chain reaction. Results: +, present; –, absent.
induced CR3-dependent neutrophil adhesion and expression of a CD18 neoepitope. This reaction was inhibited by tyrosine kinase inhibitors and by the sulfhydryl inhibitor phenylarsine oxide. These same inhibitors also blocked the neutrophil adhesion and the neoepitope expression induced by physiologic activators, as did free radical scavengers and diphenylene iodonium (DPI, an inhibitor of flavoprotein oxidoreductases). However, this oxidative thiolation step in the tyrosine kinase-dependent activation of b2 integrins is not mediated by the leukocyte NADPH oxidase, because the adhesion and neoepitope expression upon activation was normal in CGD neutrophils and also in these cells inhibited by scavengers and DPI. Thus, this provides indirect evidence that neutrophils contain an alternative oxidase involved in signal transduction.
8. Conclusions The phagocyte NADPH oxidase is a very powerful enzyme that is able to generate an enormous amount of ROS. It is indispensable for a proper defense against pathogenic microbes, as the clinical consequences of its dysfunction prove. However, the activity of this enzyme must be carefully regulated, in order to prevent unwanted harm to the host tissue. Therefore, the enzyme is composed of several subunits that are located in different parts of resting neutrophils. Upon activation of these cells, e.g. by binding of microbes to receptors on the neutrophil, these subunits are brought together and assembled into an active enzyme. This process starts with phosphorylation of cytosolic oxidase components, which introduces conformational changes that allow protein–protein interactions between the cytosolic and the
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membrane-bound oxidase components (charge mediated and SH3-proline mediated), protein–lipid interactions (through PX domains binding to phosphoinositides) and interactions with small GTPases. Together these changes result in assembly of an active NADPH oxidase in the membrane of the cell. Additional interactions with cytoskeletal proteins lead to proper positioning of the enzyme in the membrane of the phagosome that contains the ingested microbe. Probably, the products of this enzyme cooperate with proteases to kill the microbes, but a discussion is ongoing regarding the importance of each of these elements. Many microbe species have found ways of evading this antimicrobial defense system, but fortunately there is sufficient redundancy to cope with almost any microorganism. Finally, a number of homologous NADPH oxidases have been identified in various cell types, with as yet unknown function. Increased knowledge about these systems may lead to better treatment and perhaps prevention of diseases such as atherosclerosis. References [1]
D. Roos, J.T. Curnutte, Chronic Granulomatous Disease, in: H.D. Ochs, C.I.E. Smith, J.M. Puck (Eds.), Primary Immunodeficiency Diseases. A Molecular and Genetic Approach, Oxford University Press, New York, Oxford, 1999, pp. 353–374. [2] E.P. Reeves, H. Lu, H.L. Jacobs, C.G. Messina, S. Bolsover, G. Gabella, E.O. Potma, A. Warley, J. Roes, A.W. Segal, Killing activity of neutrophils is mediated through activation of proteases by K+ flux, Nature 416 (2002) 291–297. [3] D.R. Ambruso, C. Knall, A.N. Abell, J. Panepinto, A. Kurkchubasche, G. Thurman, C. Gonzalez-Aller, A. Hiester, M. de Boer, R.J. Harbeck, R. Oyer, G.L. Johnson, D. Roos, Human neutrophil immunodeficiency syndrome is associated with an inhibitory Rac2 mutation, Proc. Natl. Acad. Sci. USA 97 (2000) 4654–4659. [4] A. Abo, E. Pick, A. Hall, N.N. Totty, C.G. Teahan, A.W. Segal, Activation of the NADPH oxidase involves the small GTP-binding protein p21rac1, Nature 353 (1991) 668–670. [5] U.G. Knaus, P.G. Heyworth, T. Evans, J.T. Curnutte, G.M. Bokoch, Regulation of phagocyte oxygen radical production by the GTPbinding protein Rac2, Science 254 (1991) 1512–1515. [6] H.C. Welch, W.J. Codwell, C.D. Ellson, G.J. Ferguson, S.R. Andrews, H. Erdjument-Bromage, P. Tempst, P.T. Hawkins, L.R. Stephens, P-Rex1, a PtdIns(3,4,5)P3- and Gbc-regulated guanine-nucleotide exchange factor for Rac, Cell 108 (2002) 809–821. [7] T.G. Gabig, C.D. Crean, P.L. Mantel, R. Rosli, Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation, Blood 85 (1995) 804–811. [8] M. Tamura, T. Kai, S. Tsunawaki, J.D. Lambeth, K. Kameda, Direct interaction of actin with p47phox of neutrophil NADPH oxidase, Biochem. Biophys. Res. Commun. 276 (2000) 1186–1190. [9] R. Dana, T.L. Leto, H.L. Malech, R. Levy, Essential requirement of cytosolic phospholipase A2 for activation of the phagocyte NADPH oxidase, J. Biol. Chem. 273 (1998) 441–445. [10] P.G. Heyworth, J.T. Curnutte, J. Rae, J.D. Noack, D. Roos, E. van Koppen, A.R. Cross, Hematologically important mutations: X-linked chronic granulomatous disease (second update), Blood Cells Mol. Dis. 27 (2001) 16–26. [11] S. Suzuki, A. Kumatori, I.A. Haagen, F. Yoshito, M.A. Sadat, L.J. Hao, Y. Tsuji, D. Roos, M. Nakamura, PU.1 as an essential activator for the expression of gp91phox gene in human peripheral neutrophils, monocytes, and B lymphocytes, Proc. Natl. Acad. Sci. USA 95 (1998) 6085–6090. [12] D. Yang, S. Suzuki, L.J. Hao, Y. Fujii, A. Yamauchi, M. Yamamoto, M. Nakamura, A. Kumatori, Eosinophil-specific regulation of gp91phox gene expression by transcription factors GATA-1 and GATA-2, J. Biol. Chem. 275 (2000) 9425–9432.
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[13] A.R. Cross, D. Noack, J. Rae, J.T. Curnutte, P.G. Heyworth, Hematologically important mutations: the autosomal recessive forms of chronic granulomatous disease (first update), Blood Cells Mol. Dis. 26 (2000) 561–565. [14] J.H.W. Leusen, A. de Klein, P.M. Hilarius, A. Åhlin, J. Palmblad, C.I.E. Smith, D. Diekmann, A. Hall, A.J. Verhoeven, D. Roos, Disturbed interaction of p21-rac with mutated p67phox causes chronic granulomatous disease, J. Exp. Med. 184 (1996) 1243–1249. [15] C.B. Foster, T. Lehrnbecher, F. Mol, S.M. Steinberg, D.J. Venzon, T.J. Walsh, D. Noack, J. Rae, J.A. Winkelstein, J.T. Curnutte, S.J. Chanock, Host defense molecule polymorphisms influence the risk for immune-mediated complications in chronic granulomatous disease, J. Clin. Invest. 102 (1998) 2146–2155. [16] A.W. Segal, M. Geisow, R. Garcia, A. Harper, R. Miller, The respiratory burst of phagocytic cells is associated with a rise in vacuolar pH, Nature 290 (1991) 406–409. [17] L.M. Henderson, R.W. Meech, Proton conduction through gp91phoxG, J. Gen. Physiol. 120 (2002) 759–765. [18] D. Roos, C.C. Winterbourn, Lethal weapons, Science 296 (2002) 669–671. [19] S. Ehrt, M.U. Shiloh, J. Ruan, M. Choi, S. Gunzburg, C. Nathan, Q.-W. Xie, L.W. Riley, A novel antioxidant gene from Mycobacterium tuberculosisG, J. Exp. Med. 186 (1997) 1885. [20] A. Vazquez-Torres, Y. Xu, J. Jones-Carson, D.W. Holden, S.M. Lucia, M.C. Dinauer, P. Mastroeni, F.C. Fang, Salmonella pathogenicity island 2-dependent evasion of the phagocyte NADPH oxidase, Science 287 (2000) 1655–1658. [21] A. Gallois, J.R. Klein, L.A. Allen, B.D. Jones, W.M. Nauseef, Salmonella pathogenicity island 2-encoded type III secretion system mediates exclusion of NADPH oxidase assembly from phagosomal membrane, J. Immunol. 166 (2001) 5741–5748. [22] J.A. Carlyon, W.-T. Chan, J. Galán, D. Roos, E. Fikrig, Repression of rac2 mRNA expression by Anaplasma phagocytophila is essential to the inhibition of superoxide production and bacterial proliferation, J. Immunol. 169 (2002) 7009–7018. [23] L. van Heerebeek, C. Meischl, W. Stooker, C.J.L.M. Meijer, H.W.M. Niessen, D. Roos, NADPH oxidase(s): new source(s) of reactive oxygen species in the vascular system? J. Clin. Pathol. 55 (2002) 561–568. [24] B. Meier, A.J. Jesaitis, A. Emmendörffer, J. Roesler, M.T. Quinn, The cytochrome b558 molecules involved in the fibroblast and polymorphonuclear leukocyte superoxide-generating NADPH oxidase systems are structurally and genetically distinct, Biochem. J. 289 (1993) 481–486. [25] Y.A. Suh, R.S. Arnold, B. Lassegue, J. Shi, X. Xu, D. Sorescu, A.B. Chung, K.K. Griendling, J.D. Lambeth, Cell transformation by the superoxide-generating oxidase Mox1, Nature 401 (1999) 79–82. [26] M. Geiszt, J.B. Kopp, P. Varnai, T.L. Leto, Identification of renox, an NAD(P)H oxidase in the kidney, Proc. Natl. Acad. Sci. USA 97 (2000) 8010–8014. [27] B. Banfi, R.A. Clark, K. Steger, K.-H. Krause, Two novel proteins activate superoxide generation by the NADPH oxidase NOX1, J. Biol. Chem. 278 (2003) 3510–3513. [28] M. Geiszt, K. Lekström, J. Witta, T.L. Leto, Proteins homologous to p47phox and p67phox support superoxide production by NAD(P)H oxidase 1 in colon epithelial cells, J. Biol. Chem. 278 (2003) 20006–20012. [29] X. de Deken, D. Wang, M.C. Many, S. Costagliola, F. Libert, G. Vassart, J.E. Dumont, F. Miot, Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family, J. Biol. Chem. 275 (2000) 23227–23233. [30] E. Blouin, L. Halbwachs-Mecarelli, P. Rieu, Redox regulation of b2-integrin CD11b/CD18 activation, Eur. J. Immunol. 29 (1999) 3419–3431. [31] C. Meischl, D. Roos, The molecular basis of chronic granulomatous disease, Springer Semin. Immunopathol. 19 (1998) 417–434.
Forum in immunology
Oxidative killing of microbes by neutrophils Dirk Roos a,*, Robin van Bruggen a,b, Christof Meischl a,1 a
Sanquin Research at CLB, and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands b Emma Children’s Hospital, Academic Medical Centre, University of Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands
Abstract Neutrophils and other phagocytic leukocytes contain a phagocyte NADPH oxidase enzyme that generates superoxide after cell activation. Reactive oxygen species derived from superoxide, together with proteases liberated from the granules, are used to kill ingested microbes. Dysfunction of the phagocyte NADPH oxidase results in chronic granulomatous disease, with life-threatening infections. © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Neutrophils; Microbes; Killing; NADPH oxidase; Chronic granulomatous disease
1. Introduction Neutrophils fulfill their role as effector cells mainly by internalizing (phagocytosing) pathogens and exposing them to two destructive principles: reactive oxygen species (ROS) generated by an enzyme called the phagocyte NADPH oxidase 2 and hydrolytic granule proteins. The importance of these systems is exemplified by the clinical symptoms displayed by chronic granulomatous disease (CGD) patients with a dysfunctional NADPH oxidase in their phagocytes [1] and by mouse models in which elastase and cathepsin G have been knocked out [2]. Given their potential for destruction, it is not surprising that neutrophils and the organism as a whole have developed tight control and safety mechanisms to minimize the risk of neutrophil-inflicted damage to the organism. Examples of these control and safety mechanisms are the tight regulation of the activation of the NADPH oxidase and the high concentrations of proteinase inhibitors in plasma and tissue fluids. However, the protection thus obtained is not absolute, and well-known examples of disease states accom* Corresponding author. Tel.: +31-20-51233377; fax: +31-20-5123474. E-mail address: [email protected] (D. Roos). 1 Present address: Department of Pathology, Free University Medical Centre, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. 2 The NADPH oxidase enzyme present in phagocytic leukocytes (neutrophils, eosinophils, monocytes/macrophages) and in Epstein–Barr virus (EBV)-transformed B lymphocytes, which is dysfunctional in patients with chronic granulomatous disease, is called the “phagocyte NADPH oxidase” in this review, to distinguish it from similar, homologous systems in other tissues (see Section 7). © 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. doi:10.1016/j.micinf.2003.09.009
panied by tissue damage due to neutrophil-derived products include gout, chronic obstructive pulmonary disease, rheumatoid arthritis, autoimmune vasculitis and some forms of glomerulonephritis. 2. The phagocyte NADPH oxidase The phagocyte NADPH oxidase is a multicomponent enzyme with a redox center that transfers electrons from cytoplasmic NADPH onto extracellular (or intraphagosomal) molecular oxygen, thereby generating superoxide (Fig. 1). The overall reaction catalyzed by this enzyme is: NADPH + 2O2 → NADP+ + 2O2– + H+ The electron transfer from NADPH to oxygen is a multistep process, during which the electrons are transported sequentially along several moieties of the oxidase: NADPH → FAD → 2 Heme → 2O2 Although FAD and the two heme groups are part of the redox center of the enzyme, cytochrome b558, NADPH cannot bind to this protein unless the complete enzyme has been assembled during activation, and only then can electron transfer actually take place. Cytochrome b558, a flavo-hemeprotein, is composed of two subunits, gp91phox and p22phox, in a 1:1 stoichiometry. Incorporated in the membranes of specific granules and secretory vesicles in resting cells, cytochrome b558 becomes expressed on the membrane of the phagolysosome and on the cell surface when the granules/vesicles fuse with those larger
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Fig. 1. Schematic illustration of the electron transfer mechanism of the phagocyte NADPH oxidase. After assembly of the NADPH oxidase complex, NADPH from the cytosol can bind to the enzyme and donate its electrons. These electrons are then transmitted via FAD and two heme groups to molecular oxygen on the outside of the plasma membrane, thus generating superoxide in either the phagosome or in the extracellular environment. Reproduced with permission by Springer-Verlag Heidelberg from Ref. [31].
membrane systems during cell activation (Fig. 2). The stimulus for this activation is the binding of opsonized microorganisms or high concentrations of chemoattractants to phagocyte surface receptors. As part of this activation, the enzyme’s three cytosolic components, p47phox, p67phox and p40phox, as well as a low-molecular-weight GTP-binding protein, translocate to the cytochrome b558 in the membrane to form there the complete and active form of the NADPH oxidase (Fig. 2). A defect in any one of the four components, gp91phox, p22phox, p47phox or p67phox, abolishes (or reduces) the activity of the oxidase and leads to CGD. Defects in the enzyme component p40phox are not known. Recently, a human immunodeficiency syndrome with impaired neutrophil chemotaxis, polarization, azurophilic granule secretion and superoxide production has been shown to be caused by a dominantly negative mutation in RAC2 [3].
2.1. Phagocyte NADPH oxidase components 2.1.1. gp91phox The CYBB gene that codes for gp91phox is located on the X chromosome (Xp21.1), has a length of 30 kb and comprises 13 exons. The translation product, a protein of 570 amino acids, needs for its further maturation and stabilization the presence of p22phox. Thus, abolished expression of one protein automatically leads to simultaneous absence of the other. Posttranslational modifications are the glycosylation of three of its five potential N-linked glycosylation sites. The N-terminal half of the mature protein contains six hydrophobic, probably membrane-spanning domains, while the hydrophilic C-terminal part is thought to represent the cytosolic side of the protein. The hydrophobic part contains the heme moieties and is probably also involved in the interaction with
Fig. 2. Activation of the phagocyte NADPH oxidase. Assembly of the enzyme and phagosome formation are concomitant processes. Translocation of the cytosolic oxidase components is initiated by serine phosphorylation in p47phox and controlled by small Rho-like GTPases (Rac1, Rac2, Rap1A). This translocation leads to a conformational change in gp91phox that permits NADPH binding, thus activating the NADPH oxidase enzyme. Reproduced with permission by Springer-Verlag Heidelberg from Ref. [31].
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p22phox. For the C-terminal part, a three-dimensional model has been deduced from sequence homology with the ferridoxin NADP+ reductase flavoenzyme family. This part of the protein contains one binding site for NADPH and the FADbinding site. In the inactive state of the enzyme, the NADPHbinding site is probably covered by a loop of 20 amino acids. 2.1.2. p22phox p22phox is encoded by the gene CYBA, which is located on chromosome 16q24 and spans 8.5 kb and six exons. The resulting protein of 195 amino acids has one proline-rich region involved in interaction with SH3 (src homology region 3) domains. 2.1.3. p47phox NCF1, the gene on chromosome 7q11.23 coding for p47phox, has a length of 15 kb, encompassing 11 exons. Its product, a protein of 390 amino acids, contains nine serine phosphorylation sites, two SH3 domains, one PX (Phox homology) domain involved in targeting membranes by binding to phosphoinositides, and one proline-rich region. 2.1.4. p67phox NCF2, on chromosome 1q25 codes for p67phox, is 40 kb long and comprises 16 exons. p67phox itself, with its 526 amino acids, possesses a (higher affinity) binding site for NADPH, four tetratricopeptide repeat (TPR) motifs involved in binding of Rac1 or Rac2, two SH3 domains and one proline-rich region. 2.1.5. p40phox Very little is known yet about p40phox, a protein of 339 amino acids. Its gene, NCF4, spans 18 kb and 10 exons and is located on chromosome 22q13.1. The protein contains one SH3 and one PX domain. It probably plays a role both in stabilization of the p47phox/p67phox complex in the cytosol and in facilitating membrane recruitment of this complex during NADPH oxidase activation. 2.2. Activation of the enzyme Responding to as yet only partially unravelled upstream events that transmit the signals originating from the cell surface receptors, the activation of the NADPH oxidase itself seems to be initiated by a change in the phosphorylation of p47phox. The different protein kinases implicated in this process apparently phosphorylate different groups of serines, with each different state of phosphorylation corresponding to a different three-dimensional conformation of the protein. This process disrupts the cytosolic complex of p47phox/ p67phox/p40phox in the resting cell, exposing until then inaccessible SH3 and proline-rich domains, as well as PX domains. This change results in translocation of these three proteins to the membrane, where they associate with cytochrome b558 (Fig. 2). The interactions of the cytosolic components amongst each other and with the cytochrome are mediated by SH3 domains binding specifically to certain proline-rich
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regions, while the interactions of the cytosolic oxidase components with the membrane are PX-domain mediated. In the membrane-associated complex, p47phox appears to stabilize the interaction of p67phox with the cytochrome. p67phox with its high-affinity binding site for NADPH, on the other hand, could bind to gp91phox, which contains a lower affinity NADPH-binding site, to form the catalytically efficient binding site of the active enzyme. The loop of 20 amino acids that covers the NADPH-binding site in gp91phox in the resting state is thought to move away, as a result either of the complex formation or of an independent control mechanism. NADPH then binds to the completely assembled oxidase, electron transfer will start and superoxide generation begins (Fig. 1). Both the assembly of the enzyme and the electron flow itself are subject to regulating and modifying influences of three low-molecular-weight GTP-binding proteins, Rac1, Rac2 and Rap1A [4,5]. Functioning as molecular switches in signaling cascades, with an inactive GDP-bound and an active GTPbound state, these Rho-like GTPases themselves are again under the control of guanine-nucleotide exchange factors (GEFs) and GTPase-activating (GAP) proteins. In neutrophils, one Rac-activating GEF has been identified: P-Rex1, which is synergistically activated by phosphoinositides and bc subunits of trimeric G proteins associated with seven-span membrane receptors [6]. This indicates that P-Rex1 is under direct control of ligand (e.g. chemokine) binding to these receptors. Upon activation of Rac, these GTPases are liberated from Rho-GDI (GDP dissociation inhibitor), and as a result, a conserved carboxyl-terminal 15-carbon farnesyl group becomes exposed that mediates association of Rac with the membrane and thus with the cytochrome. Rac1 (expressed predominantly in macrophages) and Rac2 (expressed predominantly in neutrophils) interact with two of the TPR domains of p67phox. In the activated oxidase complex, Rac participates in and modulates the electron transfer steps from NADPH to FAD, heme and onto molecular oxygen. GTPase-activating proteins, finally, seem to play a role in the deactivation process of the NADPH oxidase. Few data are as yet available about the role of Rap1A in the activation of the oxidase. Rap1A associates with the NADPH oxidase in a phosphorylation-dependent manner, and clearly is involved in the regulation process, as both constitutively active or inactive mutants decrease or inhibit the respiratory burst, and overexpression of the wild-type GTP-binding protein enhances O2– production [7]. Rap1A itself is activated after stimulation of neutrophils with a variety of stimuli, including fMet–Leu–Phe (fMLP), platelet-activating factor (PAF), granulocyte-macrophage colony-stimulating factor (GMCSF), and IgG-coated particles, involving both phospholipase C-dependent and -independent pathways. Furthermore, Rap1A seems to activate protein kinase C and may, therefore, also have a more indirect role in oxidase regulation. Although it is becoming increasingly clear that interactions with other proteins—possibly in the form of a macromolecular complex—and with the cytoskeleton [8] must play an important role in the regulation and the localization of the
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oxidase activity, almost nothing is known yet about this cellular background of NADPH oxidase regulation. In addition, arachidonic acid, liberated by cytosolic phospholipase A2 from plasma membrane phopholipids, seems to play an essential role in oxidase activation [9]. Whether arachidonic acid itself exerts this effect on one or more of the oxidase components or whether the membrane lysophospholipids create a microenvironment suitable for oxidase complex formation is unknown. The impressive complexity of the activation of the enzyme and its intricate control mechanisms point to the importance of a tightly controlled, place- and time-restricted release of free oxygen radicals, since an uncontrolled release of these products would have devastating effects for the affected individual. This complexity, on the other hand, is the explanation for the fact that defects in different genes can lead to the same cellular dysfunction and disease. 3. Chronic granulomatous disease 3.1. Clinical presentation CGD is a rare disease, with an estimated incidence of 1:250,000, all ethnic groups being equally affected. The most common form of the syndrome is X-linked, and the overwhelming majority of these patients is, obviously, male; in the autosomal forms, no sex preference is discernible. CGD presents early in life in the form of acute or chronic infections, two-thirds of the patients showing the first symptoms already in their first year of life [1]. As a general rule, the organs that represent the individual’s border against the outside world, or the lymph stations downstream of those organs, are most frequently affected: lungs, skin and gastrointestinal tract. The isolated pathogens are usually catalase positive and/or relatively resistant to the other, non-oxidative killing mechanisms of the phagocytes. Catalase degrades hydrogen peroxide (H2O2), which is also produced in small amounts by the microbes themselves, and thus deprives the phagocytes of the possibility to use this microbe-generated oxygen metabolite for killing. The microorganisms most frequently found in CGD patients are Staphylococcus aureus, various Aspergillus species, enteric Gram-negative bacteria (including Serratia marcescens and various Salmonella species) and Burkholderia cepacia. Frequently encountered infections are (in descending order of frequency) pneumonia, cutaneous abscesses and suppurative lymphadenitis, hepatic and perihepatic abscesses and osteomyelitis. While the above affections normally represent acute disease states, CGD—as the name implies—is typically characterized by a chronic struggle of the immune system with the pathogens. The granulomas that can be found in a large variety of organs and to which CGD owes its name are the result of chronic inflammatory cell reactions, involving mainly lymphocytes and histiocytes. Clinically, the granulomas can become symptomatic by pain or signs of obstruction of the gastrointestinal or urinary tract. Other manifestations
of chronic inflammation are the inflammatory bowel disease of CGD, which closely resembles Crohn’s disease, an eczematoid dermatitis and, very rarely, discoid or, even more rarely, systemic lupus erythematosus. 3.2. Molecular defects in CGD 3.2.1. Defects in gp91phox All possible types of mutation, except gene conversions, have been found in CYBB, with single-nucleotide substitutions accounting for 65% of the defects, and deletions and insertions for the remaining 35%. Very large deletions, extending over other coding genes localized on the X chromosome, can result in various clinical entities, such as Duchenne muscular dystrophy, retinitis pigmentosa or McLeod’s syndrome, being associated with CGD. In a multicenter review of the mutations found in 261 X-linked CGD kindreds, 65% of these mutations were found to be family specific, with the other 35% being clustered around a few hotspots, mainly around CpG sequences [1]. The large majority of X-linked mutations in CGD lead to a complete lack of gp91phox, due to instability of the mRNA or of the translated protein. These are called X910 variants, to differentiate them from the (few) cases with reduced or normal protein expression, called X91– and X91+, respectively. In the cases of X91–, the reduced protein expression is accompanied by a roughly proportional decrease in superoxide production, whereas the X91+ variants express normal amounts of a non-functional protein. While being clinically indistinguishable from the X910 variants, the cases of X91+ CGD are of great interest for understanding the working mechanism of the oxidase, because they allow analysis of how different defects block various steps in the activation process or in the electron transport. The 15 X91+ mutations known so far [10] have contributed in this way to our knowledge of gp91phox, by identifying regions important for the binding of one of the heme groups, NADPH or the cytosolic oxidase components. Five families have been found with single nucleotide substitutions in the 5′ promoter region of CYBB. These mutations are clustered in the region around bp-55, and each ablates a binding site recognized by the Ets transcription factors PU.1 and Elf-1 [11]. In three of these families, with mutations at –52 or –53, the eosinophils still expressed gp91phox and oxidase activity, whereas the neutrophils, monocytes and EBV-transformed B lymphocytes were defective; this has led to the characterization of eosinophilspecific regulation of gp91phox gene expression by the transcription factors GATA-1 and GATA-2 [12]. 3.2.2. Defects in p22phox About 5% of the cases of CGD are caused by defects in p22phox. In the 34 families with A22-CGD investigated so far, 27 different mutations were found in the 68 alleles involved [13]. The only A22+-mutation known, a substitution of glutamic acid for one of the prolines in its proline-rich region, apparently destroys the interaction with the SH3
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domain of p47phox, thereby interrupting the activation of the enzyme. 3.2.3. Defects in p47phox Mutations in NCF1 account for about 30% of the cases of CGD. In strong contrast with the variation in the mutations found in the other subtypes of CGD, only a few different mutations have been reported in A47 CGD to date. In 52 unrelated patients described, 41 were homozygotes and eight compound heterozygotes for a dinucleotide deletion in the first four nucleotides of exon 2 (GTGT → GT) [13]; overall, 11 different mutations have been found in this patient group. This situation has long remained unexplained, but the finding that most wild-type alleles contain two pseudogenes—highly homologous but non-functional gene copies of p47phox—in addition to NCF1, has led to the realization that recombination events between NCF1 and a pseudogene cause the extremely high uniformity of mutations found in A47 CGD. 3.2.4. Defects in p67phox With around 5% of the described cases, A67 CGD also represents a rare subtype of the disease. In the 24 A67 CGD patients characterized thus far, 23 different mutations were found among the 48 alleles [13]. While the level of mRNA is usually normal, no protein expression has been found in A67 CGD, with the exception of one A67+ patient, whose p67phox protein is apparently non-functional due to the deletion of one amino acid. This deletion causes a strongly diminished binding of Rac, and thereby a disturbance in NADPH oxidase activation [14]. 3.2.5. Defects in p40phox There are no known defects in this protein. 3.3. Correlations between genotype and phenotype in CGD In general, the X910 and X91+ subforms of CGD present with similar clinical severity. It might be expected, on the other hand, that the severity of the clinical symptoms in X91– CGD correlates with the amount of residual superoxide production (3–30%) found in these patients’ phagocytes. This is, while often true, not a dependable rule. Probably, variabilities in other host-defense systems play an important role here, as borne out by a study that examined the role of host-defense molecule polymorphisms in determining immune-mediated complications in CGD. This study demonstrated, for example, strong associations of myeloperoxidase and mannosebinding lectin genotypes with gastrointestinal complications and autoimmune/rheumatologic disorders, respectively [15]. Other such associations quite certainly exist and determine cumulatively the overall phenotype. Autosomal CGD, as borne out by a few clinical studies, seems to follow in general a more benign clinical course [1]. Given the observations that p47phox, under certain in vitro conditions, seems not to be essential for oxidase activity, it might be speculated that some residual H2O2 generation is also possible in the in vivo situation.
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4. Function of the phagocyte NADPH oxidase It is clear that the defect in killing of microbes by neutrophils from patients with CGD results from the inability of these cells to generate superoxide. However, superoxide has a low bactericidal potency, so it is probably not superoxide itself that kills the microbes. Within the phagosome, superoxide is—spontaneously or enzymatically—converted into H2O2, which may then react with superoxide to generate hydroxyl radicals and singlet oxygen, both highly reactive and toxic compounds. Superoxide can also react with nitrogen oxide (NO), generated by inducible NO synthase, to yield peroxynitrite, a very reactive nitrogen intermediate. There are even indications that singlet oxygen may be converted into a compound similar to ozone (O3) in a reaction catalyzed by antibodies bound to microbes or neutrophils. H2O2 may also, together with chloride, be used as a substrate by myeloperoxidase released from azurophil granules to generate hypochlorous acid (also known as bleach), a very toxic compound for almost all microbes (Fig. 3). Subsequently, the short-lived hypochlorous acid can react with secondary amines to form secondary chloramines, which are as microbicidal as hypochlorous acid but much more stable. Is this then the way in which neutrophils kill so very efficiently the large array of microorganisms that we encounter? There is at present a debate in the literature whether this is really the case. Doubts about this concept have arisen from a number of observations. One is the fact that deficiency of myeloperoxidase is a common event but seldom leads to serious defects in the killing of microbes. Another is the observation that double knock-out mice for elastase and cathepsin G have a defect in bacterial killing similar to that of mice with a defect in the phagocyte NADPH oxidase [2]. This latter observation brought Tony Segal in London (UK) to the idea that the oxidase might be involved in the liberation of lysosomal proteases in the phagosome. Some 20 years ago already, Segal noticed that the pH in the phagosome initially increases to a value around pH 8 shortly after phagosome formation, despite the fusion of the phagosome with the acidic azurophil granules [16]. Only after about 30 min, does the intraphagosomal pH drop to values below 7. In contrast, in CGD neutrophils, the pH rapidly drops to values around 6.5. In the years to follow, it became clear that the leukocyte NADPH oxidase not only transfers electrons from NADPH in the cytosol to oxygen in the phagosome but also transfers protons from the cytosol to the phagosome, to compensate for this charge separation [17]. However, if this proton influx into the phagosome were equal to the electron influx, the pH would remain neutral, or decrease, due to fusion of the granules with the phagosome (Fig. 3). In fact, the pH increases, indicating that the proton influx is insufficient to compensate for the electron influx. Recently, Segal’s group published evidence that part of the charge compensation is due to influx of potassium ions instead of protons [2]. These investigators also found that these potassium ions are instrumental in liberating proteases such as elastase and cathepsin
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D. Roos et al. / Microbes and Infection 5 (2003) 1307–1315
Fig. 3. Within the phagosome. In the small space between an ingested bacterium (shaded area) and the membrane of the phagosome, a number of chemical reactions take place. Molecular oxygen (O2) is reduced to superoxide (O2–) by electrons pumped into the phagosome by the phagocyte NADPH oxidase. This charge transfer is compensated by an influx of protons (H+) or other cations. The protons are used to reduce superoxide to H2O2, which can be degraded to oxygen and water in a catalase-dependent reaction. Alternatively, H2O2 can combine with chloride (Cl–) to form hypochlorous acid (HOCl) in a reaction catalyzed by myeloperoxidase (MPO). If all electrons pumped into the phagosome are compensated by proton influx, the pH in the phagosome will remain neutral. However, the pH does in fact rise to about pH 8, despite the release of acid contents from granules in the cytoplasm that fuse with the phagosome [16]. This indicates that other cations, such as potassium ions (K+), may enter the phagosome instead of protons [2]. If there is an influx of K+, these cations then mediate solubilization of proteases that are bound to the proteoglycan matrix of the granules. Reproduced with permission from Ref. [18].
G from their acidic proteoglycan matrix in the granules. Thus, these observations explain the increase in intraphagosomal pH to values optimal for protease action and indicate that the NADPH oxidase, rather than killing microorganisms by its oxidative products, acts by liberating lysosomal proteases. The debate now centers around the question whether the oxidative products do have a role for themselves in the microbial killing process or not [18]. A strong argument in favor of this concept is the observation that CGD neutrophils show enhanced killing of microorganisms when coingesting latex particles coated with glucose oxidase (which generates H2O2). In addition, bacteria in which catalase or superoxide dismutase have been inactivated are killed much more easily by neutrophils or by a cell-free, O2–- or H2O2-generating system than wild-type bacteria. Moreover, the killing of bacteria by H2O2 in a cell-free system is dramatically enhanced by myeloperoxidase, and myeloperoxidase-deficient mice succumb to bacterial challenge. All this evidence points to a direct contribution of ROS to the killing process. Release of proteases from the granules can also take place in the absence of oxidase activity or even at low potassium concentrations, as indicated by the release of these enzymes into the extracellular environment and by the killing of some bacterial strains by neutrophils under anaerobic conditions. Most likely, therefore, the leukocyte NADPH oxidase induces microbial killing both in a direct way (via oxidative products) and in an indirect way (via liberation of proteases).
5. Escape of microorganisms from oxidative killing Microorganisms have developed several mechanisms to escape from the action of the phagocyte NADPH oxidase. One of these is a strong defense against the toxic oxygen metabolites, such as a high expression and even excretion of superoxide dismutase, increased expression of catalase or other, as yet unidentified defense systems. For instance, Mycobacterium tuberculosis contains several noxR genes that confer resistance against both H2O2 and reactive nitrogen intermediates when expressed in heterologous bacteria [19]. The mode of action of the proteins encoded by these genes is unknown. Recently, we have identified a Salmonella typhimurium variant with decreased resistance against H2O2 due to the inactivation of Sspj, a pyrroloquinoline quinone (PQQ)-containing protein. Probably, this protein is involved in redox reactions leading to breakdown of H2O2. Salmonella species induce their own internalization into host cells by means of a set of proteins encoded by the salmonella pathogenicity island 1 (Spi-1). These proteins form a type-III secretion system for the introduction of bacterial proteins into the cytosol of the target cell. Spi-1 also harbors genes that encode proteins that are secreted into the target cell to modulate its actin cytoskeleton, leading to the uptake of the bacteria into a membrane-bound vacuole within the target cell. Within this salmonella-containing vacuole (SCV), the bacteria are able to resist the antimicrobial response of the infected host cells, among which are neutro-
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phils. This resistance is mediated by a second group of salmonella proteins, encoded by the Spi-2 gene cluster. These proteins interfere with the activation of the NADPH oxidase by preventing the formation of an enzymatically active oxidase complex [20,21]. Our own investigations have indicated that this pathogenicity system is not complete: although wild-type Salmonella prevents oxidase activation to a large extent, a limited amount of H2O2 is still generated within the vacuoles, and this amount of H2O2 is sufficient to damage the bacteria unless they are, in addition, protected by the PQQ-containing protein Sspj (and by superoxide dismutase and catalase). In addition, we found that opsonization of Salmonella with antibodies induces a 20–50-fold higher oxidase activity than that observed with unopsonized bacteria. This shows the importance of the mode of entrance of these bacteria in the activation of the NADPH oxidase. Another way of evading oxidative killing is shown by Anaplasma phagocytophila, an obligatory intracellular bacterium with a tropism for neutrophils, transmitted by ticks and causing human granulocytic ehrlichiosis. This microbe strongly inhibits the transcription of gp91phox and Rac2, leading to the loss of expression of these proteins and the inability of the infected cells to generate superoxide, even after incubation with phorbol esters [22]. This inhibition is essential for growth and survival of the bacteria within the neutrophils. However, during initial infection of neutrophils, A. phagocytophila must also be able to circumvent the oxidative attack. Indeed, superoxide release is inhibited for more than 90% already within 30 min of incubation of human neutrophils with these bacteria. The mechanism of this last process is unknown. 6. A possible role of the phagocyte NADPH oxidase in other tissues Numerous reports have been published that describe the presence of components of the phagocyte NADPH oxidase in cell types other than leukocytes, both in humans and in animals. Most notably, in cells of the vascular system (endothelial cells, vascular smooth muscle cells and fibroblasts), these components have been detected with various techniques, ranging from Northern blotting, RT-PCR and cDNA sequencing to immunohistochemistry and Western blotting. Interest in this subject stems from observations that ROS are involved in various (patho)physiological responses of vascular cells, such as mitosis, apoptosis, migration, hypertrophy and modifications of the extracellular matrix. Indeed, ROS have been implicated in several major intracellular signal transduction pathways leading to changes in gene transcription and protein synthesis. Therefore, ROS are supposed to be involved in disease processes such as atherosclerosis and tumor progression. Table 1 summarizes the evidence for the presence of gp91phox, p22phox, p47phox and p67phox in vascular cells of animal and human sources [23]. However, many of these studies suffer from the lack of appropriate negative controls, such as material from relevant knock-out mice or from CGD patients. In particular, the possibility of cross-
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reactivity of oligonucleotides or antibodies against these components with one or more of the NADPH oxidase component homologs (see next paragraph) has to be taken into account. So far, to our knowledge, no extra-phagocyte deficiencies of NADPH oxidase components have been detected in patients with CGD. For instance, CGD fibroblasts contain a spectroscopically normal cytochrome b558 content compared with control fibroblasts and generate comparable amounts of superoxide [24]. 7. Non-phagocytic NADPH oxidases Recently, several homologs of gp91phox have been identified in cells other than phagocytes. These homologs have conserved the overall structure of six transmembrane domains with four heme-coordinating histidines in the N-terminal part of the molecule, followed by a cytosolic C-terminus that contains highly conserved binding sites for FAD and NADPH. In a new nomenclature, these proteins are now called Nox (NADPH oxidase) proteins, including gp91phox (Nox2). Mox-1 (or Nox1) mRNA is expressed in colon, prostate, uterus and vascular smooth muscle cells [25]. ROS produced by this enzyme seem to be generated intracellularly, in contrast to the situation with the phagocyte NADPH oxidase. Overexpression of Nox1 results in increased cell growth, suggesting a role for these oxygen species in signal transduction for cell growth regulation. A role of this enzyme in intracellular defense against microorganisms cannot be excluded at present. Another gp91phox homolog, called Renox or Nox4, has been detected by in situ RNA hybridization and immunohistochemistry in the renal cortex, predominantly in proximal convoluted tubule epithelial cells [26]. It has been suggested that Nox4 may participate in the oxygen-sensing mechanism that regulates the production of erythropoietin. Transfection of Nox4 into 3T3 fibroblasts resulted in enhanced superoxide production; however, these modified cells displayed substantially diminished cell growth. Some of these alternative NADPH oxidases, specifically Nox1, may interact with specific cytosolic partners for activity regulation and localization, i.e., with the recently discovered alternative members of the p47 and p67 protein families [27,28]. Finally, two similar homologs have been discovered that are characterized by an N-terminal extension containing a peroxidase homology domain, a calmodulin-like calcium-binding motif and an additional transmembrane domain [29]. RNA for these proteins, called ThOX-1 and ThOX-2 (or Duox1 and Duox2), is expressed specifically in the thyroid gland, at the apical membrane of the thyrocytes. Duox2, and perhaps also Duox1, is involved in the iodination of the thyroid hormone, because nonsense mutations in the gene for Duox2 have been identified in patients with congenital hypothyroidism. Do phagocytes also contain alternative NADPH oxidases? There is one indication that they do indeed. Blouin et al. [30] have found that the signaling pathway for b2-integrin activation involves a redox reaction, because exogenous H2O2
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Table 1 NADPH oxidase components detected by various techniques Subunit Cell type (a) In vascular cells from animals ec p22phox vsmc
Western blot
Northern blot
RT-PCR
IHC
DNA sequencing
Anti-sense
r+
r+ r+
r, p + r+
r, p + r+ r– r, rb +
r+ r+
r+
r, p +
r, p +
r+
fb gp91phox
ec
r+
Spectroscopy
r–
p47phox
p67phox
vsmc fb
c+
ec vsmc fb
b+
r, rb + p, b + r, rb +
ec vsmc fb
p+ rb +
rb +
r, rb +
(b) In human vascular cells huvec p22phox
+
+
+
ca ec vsmc fb
+ + +
+
+
huvec
+
+
vsmc fb
–
–
p47phox
huvec vsmc fb
+ +
+ +
+
p67phox
huvec vsmc fb
+ –
+ –
+
gp91phox
+ + + +
–
+
–
Table reproduced with permission by the BMJ Publishing Group from Ref. [23]. Cell type: ec, endothelial cell; fb, fibroblast; vsmc, vascular smooth muscle cell; ca ec, coronary artery endothelial cell; huvec, human umbilical vein endothelial cell. Animal species: b, bovine; c, calf; m, mouse; p, porcine; r, rat; rb, rabbit. Techniques: IHC, immunohistochemistry; RT-PCR, reverse transcriptase polymerase chain reaction. Results: +, present; –, absent.
induced CR3-dependent neutrophil adhesion and expression of a CD18 neoepitope. This reaction was inhibited by tyrosine kinase inhibitors and by the sulfhydryl inhibitor phenylarsine oxide. These same inhibitors also blocked the neutrophil adhesion and the neoepitope expression induced by physiologic activators, as did free radical scavengers and diphenylene iodonium (DPI, an inhibitor of flavoprotein oxidoreductases). However, this oxidative thiolation step in the tyrosine kinase-dependent activation of b2 integrins is not mediated by the leukocyte NADPH oxidase, because the adhesion and neoepitope expression upon activation was normal in CGD neutrophils and also in these cells inhibited by scavengers and DPI. Thus, this provides indirect evidence that neutrophils contain an alternative oxidase involved in signal transduction.
8. Conclusions The phagocyte NADPH oxidase is a very powerful enzyme that is able to generate an enormous amount of ROS. It is indispensable for a proper defense against pathogenic microbes, as the clinical consequences of its dysfunction prove. However, the activity of this enzyme must be carefully regulated, in order to prevent unwanted harm to the host tissue. Therefore, the enzyme is composed of several subunits that are located in different parts of resting neutrophils. Upon activation of these cells, e.g. by binding of microbes to receptors on the neutrophil, these subunits are brought together and assembled into an active enzyme. This process starts with phosphorylation of cytosolic oxidase components, which introduces conformational changes that allow protein–protein interactions between the cytosolic and the
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membrane-bound oxidase components (charge mediated and SH3-proline mediated), protein–lipid interactions (through PX domains binding to phosphoinositides) and interactions with small GTPases. Together these changes result in assembly of an active NADPH oxidase in the membrane of the cell. Additional interactions with cytoskeletal proteins lead to proper positioning of the enzyme in the membrane of the phagosome that contains the ingested microbe. Probably, the products of this enzyme cooperate with proteases to kill the microbes, but a discussion is ongoing regarding the importance of each of these elements. Many microbe species have found ways of evading this antimicrobial defense system, but fortunately there is sufficient redundancy to cope with almost any microorganism. Finally, a number of homologous NADPH oxidases have been identified in various cell types, with as yet unknown function. Increased knowledge about these systems may lead to better treatment and perhaps prevention of diseases such as atherosclerosis. References [1]
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