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Published online before print February 2, 2005
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Department of Medicine, University of Washington, Seattle
1 Correspondence: Department of Medicine, Box 357185, University of Washington School of Medicine, Seattle, WA 98195-7185. E-mail: seym{at}u.washington.edu
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ABSTRACT |
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Key Words: neutrophil microbicidal activity • hypochlorous acid • hydrogen peroxide • myeloperoxidase-mediated antimicrobial system • MPO-independent antimicrobial systems • MPO deficiency
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INTRODUCTION |
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In February 1957, I began a post-doctoral fellowship at The Rockefeller University (New York, NY), working in the laboratory of Reginald Archibald. This was an endocrine laboratory, and my interest at that time was in the thyroid gland, specifically in the biosynthesis of the thyroid hormones and in their mechanism of action. Thyroid hormone synthesis involves the iodination of tyrosine residues of thyroglobulin to form mono- and diiodotyrosine, which then couple to form the thyronine derivative, thyroxine or triiodothyronine. Both of these steps in thyroid hormone synthesis are catalyzed by a thyroid peroxidase. In regard to mechanism of action, it was found that phenolic hormones, namely the thyroid hormones and estrogens, had a marked stimulatory effect on some, but not all, reactions catalyzed by peroxidase [1 , 2 ]. Thus, the oxidation of epinephrine, uric acid, and ferrocytochrome C by peroxidase and hydrogen peroxide (H2O2) was strongly stimulated by the phenolic hormones, whereas the oxidation of guaiacol was not. The stimulatory activity of thyroxine and estradiol on peroxidatic reactions was lost on the blocking of the phenolic hydroxyl group of the hormones by the formation of the methyl ether, suggesting that the phenolic hormones acted as oxidation-reduction catalysts, first being oxidized by peroxidase and H2O2 to a form, probably the phenoxy radical, which was then reduced back to its original form by reaction with an electron donor, whose oxidation was thus accelerated (Fig. 1 ). In the absence of an appropriate electron donor, the phenolic hormones were further oxidized and inactivated.
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Agner [6 ], who had prepared MPO in a highly purified form in the early 1940s, had reported its presence in neutrophils at concentrations no less than 1–2% of the dry weight of the cells, and others [7 ] reported its presence at concentrations greater than 5%. Agner initially called the enzyme verdoperoxidase, as a result of its intense green color, but the name was subsequently changed to myeloperoxidase. It is what gives pus its green color. Cytochemical studies dating back to the early 1900s suggested the presence of a peroxidase in the cytoplasmic granules of mature granulocytes, and subsequent studies indicated its presence entirely in the azurophil (primary) granules of these cells. MPO synthesis is initiated in the promyelocyte stage of neutrophil development and terminates at the beginning of the myelocyte stage, at which time the MPO-containing azurophil granules are distributed to daughter cells, where they intermingle with the newly formed peroxidase-negative, specific (secondary) granules. The MPO-containing granules in the mature human neutrophil are heterogeneous by density [8 ] and by morphology [9 ]. Human monocytes also contain MPO-positive granules, although they are fewer in number than in neutrophils [10 ]. The MPO-containing granules are formed in bone marrow promonocytes, are readily apparent in mature monocytes, and are generally lost when monocytes mature into macrophages in tissues (see Atherosclerosis and Multiple sclerosis below for evidence for the presence of MPO in tissue macrophages under certain conditions). A peroxidase is also present in the cytoplasmic granules of eosinophils [11 ]; however, it differs from MPO structurally and in its function [12 ].
MPO is the product of a single gene, which has been cloned in a number of laboratories [13
14
15
16
17
18
19
]. The gene is 
11 kb in size, composed of 11 introns and 12 exons [20
], and located in the long arm of chromosome 17 in segment q12–24 [17
, 21
22
23
]. Its initial translation product is an 80-kD protein [24
], which following proteolytic removal of the 41 amino acid signal peptide, undergoes N-linked glycosylation with the incorporation of mannose-rich side-chains [25
, 26
] to generate an 89- to 90-kD enzymatically inactive apoproMPO [20
], which forms a complex in the endoplasmic reticulum with the calcium-binding proteins calreticulin and calnexin, which act as molecular chaperones [27
, 28
]. With the insertion of a heme, apoproMPO is converted to the enzymatically active proMPO [29
30
31
]. The removal of the N-terminal 125 amino acid pro-region by proteolytic cleavage results in the production of a 72- to 75-kD protein, which undergoes a second proteolytic cleavage to generate the 467 amino acid heavy (
) subunit (57 kD) and the 112 amino acid light (ß) subunit (12 kD) of MPO, which associate as a heavy-light protomer. Mature MPO has a molecular mass of 150 kD and consists of a pair of heavy-light protomers [32
33
34
34
] whose heavy subunits are linked by a disulfide bond along their long axis [32
]. The mannose-rich carbohydrate and the two hemes are covalently bound to the heavy subunit [33
34
35
]. Reduction and alkylation result in the cleavage of MPO into hemi-MPO, which consists of a single and ß protomer. It retains enzymatic and bactericidal activity [32
, 36
]. The two heavy () subunits appear to be structurally different [37
, 38
]. The X-ray crystallographic structure of canine [39
40
41
42
] and human [43
] MPO has been described.
In 1920, Graham [44 ] first reported the release of peroxidase from neutrophil cytoplasmic granules during phagocytosis as follows: "Very marked changes in the granules may readily be determined by the study of leucocytes engaged in phagocytosis, as for example, in opsonic preparations or in smears of pus. Smears from an active case of gonorrhoea are very satisfactory. When such preparations are stained with a ‘peroxidase’ reagent, interesting examples of the more or less complete disappearance of the granules from the individual cells may be obtained. While exceptions may occur, it may be stated in general that the granules disappear from the leucocytes progressively as the number of bacterial inclusions in the cell increases." In 1960, Hirsch and Cohn [45 ] described in detail the degranulation process in phagocytes, in which the membrane of the cytoplasmic granules fused with that of the developing phagosome, the common membrane then ruptured, and the contents of the granules were discharged with great force into the phagosome. Cohn and Hirsch [46 ] then isolated the granules of rabbit granulocytes and demonstrated the presence in them of a variety of hydrolases as well as an antimicrobial protein, which in 1956, Skarnes and Watson [47 ] had called leukin, and Hirsch [48 ] had called phagocytin. The concept that granule components released into the phagosome may be toxic to ingested organisms was thus born. Subsequent studies by myself [49 ] and others indicated that MPO was among the granule enzymes discharged into the phagosome during the degranulation process. MPO also can be released to the outside of the cell by leakage before complete closure of the developing phagosome or in response to stimulation by an antibody/complement-coated surface too large to be ingested. MPO is a strongly basic protein with an isoelectric point >10 [6 ] and thus binds avidly to negatively charged surfaces. It can be seen coating ingested microorganisms and when released to the outside of the cell, to biological membranes. MPO, when released, can be inactivated by products of the respiratory burst [50 , 51 ] or be cleared from the extracellular fluid by uptake by macrophages [52 , 53 ] through reaction with the mannose receptor [53 ]. Further, the uptake of microorganisms coated with extracellularly released MPO [54 ] or eosinophil peroxidase [55 56 57 ] may arm the macrophages, resulting in an associated increased destruction of the ingested organisms.
It was clear at that time (1959–60) that the process of phagocytosis and degranulation by phagocytes was associated with increased metabolic activity [58 , 59 ]. Of particular interest was the burst of oxygen consumption, which was first reported by Baldridge and Gerard [60 ] in 1933. However, it was not until 1961, when Iyer, Islam, and Quastel [61 ] reported that the respiratory burst of phagocytes was associated with the formation of H2O2, that a product of the respiratory burst was implicated in antimicrobial activity. They stated, "In the consideration of the various factors that may operate in bringing about bactericidal action during phagocytosis, the possibility that hydrogen peroxide is formed during this process must be taken into account. It is unlikely that hydrogen peroxide is wholly responsible for the non-specific bactericidal effects of phagocytosis, but it will surely be one of the responsible agents."
As the primary function of neutrophils is the phagocytosis and destruction of microorganisms, the question was posed: Is the function of MPO in neutrophils the destruction of ingested microorganisms? With a vial of green MPO in hand, I proposed to Jim Hirsch that we look at the antimicrobial properties of this enzyme. Our first studies in this regard in 1961–62 were disappointing. We mixed viable microorganisms with MPO and sublethal levels of H2O2 and found no fall in the viable cell count. It was theoretically possible that MPO, rather than being directly toxic to bacteria in the presence (or absence) of H2O2, could act indirectly by catalyzing the conversion of a nontoxic agent to a toxic one. Thus, Kojima [62 ] reported that peroxidase (source unspecified) and H2O2 can increase the germicidal effect of a number of phenols by conversion to the corresponding quinone. Iodide seemed ideal for this purpose, as it is nontoxic but is oxidized by peroxidase and H2O2 to iodine, a well-known germicidal agent. When iodide was added to the MPO-H2O2 system, the solution turned yellow as iodine was formed, and the microorganisms were killed. The concentration of iodide required, however, was considerably greater than physiologic. At this point, I moved from The Rockefeller University to the University of Washington (Seattle), and this line of study was put to one side.
My interest in the antimicrobial properties of peroxidases resurfaced a few years later in saliva. In 1934, an antilactobacillus system was described in saliva, which differed from the then-known antimicrobial systems. Its nature remained a mystery until 1959 when Zeldow reported [63 ] that this system could be divided into a heat-stable, dialyzable component and a heat-labile, nondialyzable component, each of which was ineffective alone but which when combined, was toxic to the lactobacilli. In 1962, Dogon, Kerr, and Amdur [64 ] reported that the heat-stable, dialyzable component was the thiocyanate ion, and in 1965, Luebke and I reported [65 ] that the heat-labile, nondialyzable component was the salivary peroxidase (LPO). H2O2 was an additional requirement for this system [65 , 66 ]. It was fortuitous that lactobacillus was the target organism used in the early studies, as this organism can generate H2O2 in large amounts and thus, was able to generate the H2O2 required for its own destruction when combined with LPO and thiocyanate ions. When H2O2 or a H2O2-generating system was added, toxicity was extended to microorganisms which did not generate H2O2 themselves. A similar antimicrobial system was detected in milk [67 ].
The observation at that time that MPO could replace LPO in the peroxidase-thiocyanate-H2O2 antimicrobial system [65 , 66 ] renewed interest in the possibility that MPO served an antimicrobial function in the neutrophil. In 1967, I reported that MPO, H2O2, and iodide, bromide, chloride, or thiocyanate ions formed a powerful antimicrobial system in neutrophils [68 69 70 ] (Fig. 2 ). Of particular interest was the observation that chloride, at physiologic concentrations, could meet the halide requirement of the MPO-dependent antimicrobial system, although it was ineffective in the LPO-H2O2 system. In 1967, McRipley and Sbarra [72 ] reported that a guinea pig leukocyte extract was bactericidal when combined with H2O2, and they subsequently confirmed the requirement for a halide [73 ].
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MECHANISMS OF H2O2 FORMATION FOR THE MPO SYSTEM |
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Phagocyte NADPH oxidase
Rossi and Zatti [74
] first proposed in 1964 that a NADPH oxidase was responsible for the respiratory burst of phagocytes. In 1973, Babior, Kipnes, and Curnutte [75
] reported that the initial product of the respiratory burst oxidase was the O
as follows:
 |
-generating NADPH oxidase system of phagocytes (for review, see refs. [76
77
78
79
]). A major advance was the demonstration that the NADPH oxidase could be activated in cell-free leukocyte preparations by an anionic detergent such as arachidonic acid, sodium dodecyl sulfate, or other cis-unsaturated fatty acids [80
81
82
]. The NADPH oxidase activated in this way could be separated by centrifugation into a particulate (membrane) fraction and a soluble (cytosolic) fraction, both of which were required for NADPH oxidase activity. In 1978, Segal and Jones [83
] reported the presence of a novel b-cytochrome in high concentrations in normal neutrophils as well as in monocytes, macrophages, and cosinophils [84
]. It is a heterodimer consisting of a 91-kD glycoprotein (ß subunit), designated gp91phox, and a 22-kD polypeptide ( subunit), designated p22phox. The b-cytochrome has a characteristic absorption peak at 558 nm and a low mid-point potential (–245 mV), which allows it, by oxidation-reduction, to directly reduce O2 to O. The b-cytochrome also contains a flavin (flavin adenine dinucleotide) as part of the electron transport chain and thus, has been termed flavocytochrome b558. The gene for the heavy subunit of the b-cytochrome (gp91phox) was first identified and cloned by Orkin and his colleagues [85
, 86
] by "reverse genetics" in which the gene was initially located on the X chromosome by linkage analysis and its protein product subsequently determined from the base sequence. In the resting cell, the b cytochrome is present largely in the membranes of secondary (specific) granules and secretory vesicles in the cytoplasm and is transported to the cell (or phagosomal) membrane when the leukocyte is stimulated. It is the particulate (membrane) component of the cell-free NADPH oxidase system. The soluble (cytoplasmic) components of the oxidase consist of 67 kD, 47 kD, and 40 kD proteins, designated p67phox, p47phox, and p40phox and the low molecular weight guanosine 5'-triphosphate (GTP)-binding protein rac 1 [87
] or rac 2 [88
]. Rac 2, which is a member of the Rho family of small GTPases, appears to be the predominant GTP-binding protein of human neutrophils [89
]. The development of a polyclonal antiserum, which recognized p47phox and p67phox [90
], led to the cloning and structural characterization of the cytoplasmic components of the oxidase [91
, 92
]. The NADPH oxidase is activated by the migration of the cytoplasmic components to the cell membrane to form a complex with the membrane component flavocytochrome b558. This is facilitated by the phosphorylation, largely by protein kinase C, of p47phox, which binds to the 22-kD subunit of the b-cytochrome. The dissociation of Rac from a cytoplasmic complex with an inhibitor molecule (guanosine diphosphate dissociation inhibitor) occurs, with the conversion of Rac from its guanosine 5'-diphosphate-bound inactive form to its GTP-bound active form (for a review of NADPH oxidase regulation by Rac, see ref. [93
]). The activated NADPH oxidase transports electrons from NADPH on the cytoplasmic side of the membrane to oxygen in the extracellular fluid or when the membrane is invaginated in the intraphagosomal space to form O. The NADPH-binding site, flavin and heme, required for this electron transport, appears to be present in the gp91phox component of the NADPH oxidase, with the cytosolic components serving an activating function.
In 1967 Quie et al. [94
] described a microbicidal defect in the neutrophils of patients with chronic granulomatous disease (CGD) of childhood, a condition that had been described clinically 10 years earlier by Berendes, Bridges, and Good [95
]. CGD is characterized by severe, repeated, and widespread infections, generally involving staphylococci, certain Gram-negative bacteria, and fungi and affecting a variety of tissues. Infections generally appear in infancy and often result in early death. The lesions are often characterized by granulomata with characteristic lipid-filled histiocytes and thus the name chronic granulomatous disease. In 1967, Holmes, Page, and Good [96
] reported that the neutrophil microbicidal defect in CGD was associated with the absence of respiratory burst activity, suggesting an important role for the respiratory burst in the microbial activity of phagocytes. The basis for the respiratory burst defect in CGD is mutations in components of the NADPH oxidase, namely gp9lphox, p47phox, p67phox, and p22phox (for review, see refs. [78
, 79
, 97
, 98
]). The gp91phox gene is located on the X-chromosome, and thus, CGD due to mutations in that gene (60% of cases), affects males and is generally [99
] but not always [100
] associated with the absence of flavocytochrome b558. The remaining cases of CGD are autosomal in nature and occur with equal frequency in males and females. Approximately 30% of patients with CGD have a mutation in p47phox, 5% in p67phox, and 5% in p22phox [90
, 97
, 98
, 101
]. Recently, an infant with decreased Rac2 levels (41% of normal in neutrophil lysate, 31% of normal in neutrophil cytosol), a dominant-negative Rac2 mutation, multiple neutrophil functional abnormalities, and delayed wound-healing, complicated by infection, has been described [102
103
104
].
The initial product of the respiratory burst oxidase, O, exists in equilibrium with its protonated form, the perhydroxyl radical (HO2·) The pKa of the dissociation is 4.8 so that the radical exists almost entirely as O at neutral or alkaline pH. O reacts predominantly as a reductant, where it gives up an electron and is converted back to oxygen. It can, however, also act as an oxidant, where it accepts an electron and is converted to H2O2. When two molecules interact, one is oxidized, and the other is reduced in a dismutation reaction with the formation of O2 and H2O2. This reaction can occur spontaneously or be catalyzed by superoxide dismutase (SOD). Spontaneous dismutation occurs optimally at pH 4.8, where O and HO2· are present in equal concentrations. At this pH, the rate constant for spontaneous dismutation approaches that of SOD-catalyzed dismutation. As the pH rises, and O predominates, the rate of spontaneous dismutation falls sharply, and catalysis by SOD becomes more significant. There is no evidence that leukocytic SOD is released into the phagosome; SOD, however, can be introduced there as a component of the ingested organism.
Vascular NAD(P)H oxidase
A NAD(P)H oxidase, similar but not identical to the leukocyte enzyme, is present in vascular cells [105
106
107
108
109
110
] and may serve as a source of reactive oxygen species (ROS) in or adjacent to the vessel wall. It is of interest in this regard that in a rodent model of acute inflammation in which rats are treated with lipopolysaccharide, MPO, presumably released by stimulated phagocytes in the bloodstream, can be detected on the endothelial surface, within the endothelial cells, and in the subendothelial space, where it may react with H2O2 formed by the vascular NAD(P)H oxidase to modulate nitric oxide (NO·)-dependent signaling [111
].
NOX/DUOX enzymes
The NADPH oxidase of phagocytes is the most reactive of a family of oxidases designated NOX for NADPH oxidase or DUOX for dual oxidase [112
, 113
], which contain a region with homology to the gp91phox of the phagocyte NADPH oxidase. The DUOX enzymes also contained a peroxidase domain. A NOX homologous to the phagocyte gp91phox was first described in colon epithelial cells by Lambeth and his colleagues in 1999 [114
] and was designated MOX1 (currently NOX1). In subsequent studies, this group and others [115
116
117
118
119
] have identified NOX1 as well as homologues, designated NOX3, NOX4, and NOX5, in a variety of other tissues [113
]. The phagocyte NADPH oxidase was designated NOX2. In general, ROS production by the nonphagocyte NOX enzymes is considerably less than that produced by the phagocyte NOX (NOX2). However, recently, the proteins NOX organizer 1 and NOX activator 1, with homology to the NOX2 p47phox and p67phox, respectively, have been cloned from colon epithelial cells [120
121
122
123
], and their inclusion with NOX1 greatly increases ROS production. Elevated calcium levels can also increase superoxide production by some NOX enzymes, e.g., NOX5, which contains four EF-hands [118
, 124
].
DUOX1 and 2 were first described in the thyroid gland [116 , 117 , 125 , 126 ] and were designated THOX1 and -2 [117 ]. The THOX proteins are colocalized with the thyroid peroxidase at the apical membrane of human thyroid cells [125 ], and these two enzymes thus may combine to catalyze thyroid hormone synthesis. The DUOX (THOX) enzymes by virtue of having a superoxide-generating and a peroxidase center also may serve to generate and use H2O2 for thyroid hormone synthesis, although it is not clear that the DUOX enzymes have appreciable peroxidase activity. Mutations in DUOX2 (THOX2) are associated with a loss of thyroid hormone synthesis and can lead to congenital hypothyroidism [126 ]. The DUOX1 of Caenorhabditis elegans uses its dual functionality to facilitate cuticle formation by cross-linking of tyrosine residues in extracellular proteins [121 ]. DUOX1 and DUOX2 were found in salivary gland, rectum, trachea, and bronchium, and it was proposed that these enzymes serve as the source of H2O2 for a LPO-mediated antimicrobial system at mucosal surfaces [127 ].
Soluble H2O2-generating enzymes
Certain soluble enzyme systems can form H2O2, some via a O intermediate and others, without the formation of detectable O. Thus, xanthine oxidase and amine oxidase form O and H2O2, whereas glucose oxidase forms H2O2 without an apparent O intermediate. Xanthine oxidase has been implicated as a source of ROS in ischemia-reperfusion injury [128
].
Mitochondrial metabolism
Mitochondrial electron transport systems in a variety of cell types generate small amounts of O and H2O2; however, it is not clear whether the H2O2 formed in this way can be released to the outside of the cell in sufficient amounts to be damaging to adjacent tissue.
Microbial metabolism
Certain microorganisms, namely those designated as lactic acid bacteria, generate H2O2 [129
]. These microorganisms, which include strains of streptococci, pneumococci, and lactobacilli, lack heme and thus do not use the cytochrome system (which converts oxygen to water) for terminal oxidations. Rather, flavoproteins are used, which convert oxygen to H2O2. These organisms also lack a heme catalase and thus do not efficiently degrade H2O2, which accumulates in the medium. The finding that lactobacilli can serve as a source of H2O2 for the peroxidase-mediated antimicrobial system is pertinent to the female genital tract, as the predominant organism in the normal human vagina is the lactobacillus. In one study [130
], 96% of normal women harbored H2O2-generating lactobacilli, whereas 4% harbored lactobacilli, which did not generate H2O2. Bacterial vaginosis typically occurs in sexually active women who complain of vaginal discharge, irritation, and odor. There is an associated overgrowth with Gram-negative coccobacilli, Gardnerella vaginalis, and certain anaerobes in the vagina associated with a decrease in H2O2-generating lactobacilli. In our series, only 6% of women with bacterial vaginosis harbored H2O2-producing lactobacilli, and 36% harbored lactobacilli which did not generate H2O2 [130
]. Similarly, the absence of H2O2-generating lactobacilli predisposes women to vaginal Escherichia coli colonization and urinary tract infection [131
]. H2O2-producing lactobacilli, particularly in the presence of peroxidase and a halide, are bactericidal to G. vaginalis [132
] and E. coli [133
] and are viricidal to human immunodeficiency virus type 1 (HIV-l) [134
]. Lactobacilli alone at low concentrations also are toxic to certain anaerobes through the formation of H2O2 [132
]. Peroxidase activity has been detected in vaginal fluid specimens from most women in amounts sufficient to induce a microbicidal effect in vitro [132
], and chloride is also present in human cervical mucus in excess [135
]. These findings raise the possibility that production of H2O2 by lactobacilli may represent a nonspecific host defense mechanism in the normal vagina in the presence or absence of peroxidase of leukocytic or uterine origin.
Nonoxynol-9 is a nonionic detergent with spermicidal activity that is widely used as the active ingredient in a number of vaginal contraceptive preparations. In addition, nonoxynol-9 is toxic to a variety of microorganisms, suggesting that nonoxynol-9-containing contraceptive preparations also may provide protection against certain genital infections. Epidemiological studies have provided support for this protective effect [136 ]. Paradoxically, women who use spermicides have increased vaginal colonization with E. coli [131 , 137 ] and an increased incidence of bacteriuria with this organism. E. coli are resistant to the direct toxic effect of nonoxynol-9 [138 ], whereas lactobacilli are highly sensitive. This raises the possibility that suppression of the growth of lactobacilli in the vagina by nonoxynol-9 may favor the survival of E. coli by preventing its destruction by lactobacilli-derived H2O2 in the presence or absence of peroxidase and a halide. Nonionic detergents including nonoxynol-9 form peroxides when exposed to oxygen for a prolonged period, and the peroxides so formed can be toxic to E. coli when combined with MPO and chloride [133 ].
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PRODUCTS OF THE MPO-MEDIATED ANTIMICROBIAL SYSTEM |
|---|
[146
147
148
], as well as by a number of other reducing agents [146
, 149
, 150
] with restoration of the ability to oxidize chloride to form HOCl. Kettle and Winterbourn [146
, 148
, 151
] have proposed that one of the functions of O may be to maintain MPO in an active form in the presence of excess H2O2. O may thus potentiate oxidant damage at inflammatory sites by optimizing the MPO-dependent production of HOCl, and the anti-inflammatory effect of SOD may be in part a result of the inhibition of this reaction. However, the conversion of compound II to native MPO by O is considerably slower than the comparable conversion by certain other reducing agents, e.g., ascorbic acid [143
, 152
], raising a question about the physiologic role of the O-dependent activation of MPO compound II. O can also react directly with native MPO to form compound III [142
, 153
154
155
156
], an oxyperoxidase, which like oxyhemoglobin, has oxygen attached to the heme iron [157
, 158
]. Compound III is unstable, decaying to native MPO with a half-decay time of several minutes at room temperature [157
, 159
]. Compound III also can be converted to the native enzyme by reducing agents such as ascorbic acid with the return of compound I-dependent HOCl production [152
]. Compound III, which has been detected in intact, stimulated neutrophils [154
], can react with a number of compounds, both electron donors [157
, 160
161
162
] and electron acceptors [162
], raising the possibility that it is a catalytically active form of MPO in neutrophils. The reaction of MPO with O to form compound III, however, is an order of magnitude slower than the reaction of native MPO with H2O2 [156
].
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 |
as follows:
 |
to form ·OH (Haber-Weiss reaction, superoxide-driven Fenton reaction) as follows:
 |
are formed by stimulated phagocytes, their interaction to form ·OH might be expected. Trace metal (e.g., Fe) catalysis of the Haber-Weiss reaction is required, as H2O2 and O do not interact directly at an appreciable rate. The free iron concentration in biological fluids is extremely low and would be expected to limit the formation of ·OH by the Haber-Weiss reaction. The bulk of the body’s iron is bound to protein for storage and transport or to form a catalytic center. The ability of protein-bound iron to catalyze the Haber-Weiss reaction under physiological conditions has not been clearly demonstrated.
·OH appear to be formed by the MPO-H2O2-Cl– system in neutrophils, at least in small amounts (for review, see ref. [12
]). One of the methods for the detection of highly labile free radicals is to form a relatively stable radical adduct with a spin trap, which can be detected by electron spin resonance spectroscopy. A new spin trap procedure for the detection of ·OH, in which ·OH reacts with ethanol to form the -hydroxyethyl radical, which forms a measurable adduct with the spin trap -(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN), was applied by Ramos et al. [185
] to neutrophils. This procedure was an order of magnitude more sensitive than previously used methods, and with it, ·OH formation by stimulated neutrophils could be detected. Further, evidence was presented suggesting that the formation of ·OH by stimulated neutrophils was by a MPO-dependent mechanism. They proposed that MPO catalyses the H2O2-dependent formation of HOCl, which reacts with O to form ·OH as follows:
 |
 |
, by catalase implicating H2O2, and by azide implicating MPO. Further, the reaction of purified MPO with the xanthine oxidase system, which generates O and H2O2 , resulted in the formation of ·OH in a reaction that was dependent on chloride and was inhibited by SOD, catalase, and azide. However, less than 1% of neutrophil O and H2O2 production could be accounted for by the formation of ·OH by this mechanism, raising a question about its physiological significance. ·OH is an extremely reactive radical, which will react with essentially the first molecule it meets. Thus, it would need to be formed in the immediate vicinity of the crucial target on the bacterial surface [186
]. The HCO3· radical, formed by the reaction of ·OH with CO2, may be an effective microbicide under the conditions present in the phagosome [186
].
Singlet oxygen (1O2)
In early studies, formation of 1O2 by neutrophils could not be detected using the conversion of cholesterol to 3ß-hydroxy-5-cholest-6-ene-5-hydroperoxide as a specific marker of 1O2 formation [187
, 188
] or by the use of instrumentation, which could detect the emission of delta 1O2 decay at 1270 nm [189
, 190
], leading to the suggestion that 1O2 is, at best, a minor product of the respiratory burst. However, Steinbeck et al. [191
] have provided evidence supporting the formation of 1O2 by the MPO system in neutrophils, using the conversion of 9,10 diphenylanthracene (DPA) to the DPA-endoperoxide as a specific and sensitive measure of 1O2 formation. When neutrophils ingested beads coated with DPA, up to 19% of the oxygen consumed could be accounted for by the formation of 1O2. 1O2 was also detected with this technique as a product of the MPO-H2O2-chloride system, and the mechanism for its formation was the reaction of H2O2 with HOCl/OC1– (or Cl2), a classical mechanism for the formation of 1O2 as follows:
 |
 |
Ozone (O3)
Recently, it has been proposed that antibodies, regardless of antigen specificity, can catalyze the oxidation of water by 1O2 to produce H2O2 and O3 [194
195
196
197
]. O3 has also been proposed as a product of the respiratory burst of neutrophils, using 1O2 formed by the MPO system for its formation [198
]. O3 is bactericidal, and a combination of H2O2 and O3 is more toxic to microorganisms than either alone. This thus may be an additional mechanism for the destruction of microorganisms by the MPO system of phagocytes.
The formation of O3 by stimulated phagocytes was based largely on the conversion of indigo carmine to isatic sulfonic acid, a reaction that can be induced by O3. However, O is also capable of this reaction, and the involvement of O in the conversion of indigo carmine to isatic sulfonic acid by stimulated phagocytes is suggested by the inhibitory effect of SOD and the absence of inhibition by catalase, azide, and methionine [199
].
|
EVIDENCE FOR MPO INVOLVEMENT IN NEUTROPHIL MICROBICIDAL ACTIVITY |
|---|
MPO, H2O2, and a halide form a powerful antimicrobial system
Initial studies indicated that the halide requirement could be met by iodide, bromide, or chloride [68
69
70
] or by the pseudohalide, thiocyanate [65
, 66
]. More recently, nitrite was shown to substitute for the halide in the MPO-mediated antimicrobial system in vitro [202
, 203
]. Nitrite was bactericidal at concentrations down to 10–3M when combined with MPO and a source of H2O2 [202
]. Nitrite can be formed in phagocytes as a product of the metabolism of NO·; however, it is not clear that nitrite is formed in sufficient amounts to contribute significantly to the microbicidal activity of the MPO system.
It is the prevailing view that chloride is the physiological halide, as it is present in biological fluids at concentrations considerably higher than that required. Iodide is the most effective halide on a molar basis; however, the concentration of free iodide in biological fluids is very low (<1 mg%). The iodinated hormone, thyroxine, can substitute for iodide in the cell-free MPO system [69 ], presumably, at least in part, as a result of its deiodination by the peroxidase system [204 , 205 ]. Bromide is intermediate between iodide and chloride in effectiveness and concentration; however, at levels present in plasma, chloride is preferentially used by the MPO system [206 ]. The presence of brominated compounds (e.g., 3-bromotyrosine) as well as chlorinated compounds (e.g., 3-chlorotyrosine) in the peritoneal fluid of wild-type mice with sepsis at concentrations considerably higher than those seen in peritoneal fluid of septic MPO-deficient mice supports a contribution by bromide to the MPO-mediated antimicrobial system in vivo [207 ]. Thiocyanate is readily oxidized by MPO and H2O2 [208 , 209 ] even in the presence of physiologic concentrations of chloride [208 ], suggesting that the product of thiocyanate oxidation, hypothiocyanous acid, may contribute to the antimicrobial activity of the MPO system.
MPO and H2O2 are formed or released by neutrophils at a time and place appropriate to the microbicidal act
MPO [49
] and H2O2 [210
, 211
] can be detected in the phagosome by electronmicroscopic cytochemical techniques. The oxidase responsible for O [212
] and H2O2 [211
] production was detected in low amounts on the plasma membrane of resting neutrophils and following phagocytosis, was found in considerably increased amounts in the phagosome. These findings indicate that the oxidase is membrane-associated and is internalized and activated during phagocytosis with the generation of H2O2 within the phagosome.
The formation of HOCl accounts for a high proportion of the oxygen consumed in the respiratory burst
Values of at least 28% [213
] and 72% [214
] with different stimuli have been proposed. Similarly, in one study, 40% of the H2O2 generated by zymosan-stimulated neutrophils was used for the formation of HOCl [215
]. These are minimum values, as they do not take into account the presence of proteins and other scavengers that compete for HOCl in the assay system [213
]. Jiang et al. [216
] concluded from their studies that "the neutrophil is capable of intraphagosomal generation of HOCl in sufficient quantities to kill entrapped bacteria on a time scale that is associated with bacterial death", and Hampton et al. [200
] concluded from their studies that "enough HOCl is generated in the phagosome for it to be responsible for killing."
MPO, H2O2, and a halide interact in the phagosome adjacent to the ingested organism
The production of ROS and their reactions occur in the microenvironment of the phagosome, which is complex and constantly changing [186
]. Initially, when opsonized bacteria are ingested by phagocytes, microbe-associated ligands, generally antibody and/or complement, bind to membrane receptors in a continuous and circumferential manner, as the cell membrane invaginates to form a tight phagosome, with little or no space between the microbe and phagosome membrane (zipper phenomenon [217
]). This process is associated with the activation of the NADPH oxidase and with degranulation, which releases ROS and the granule components, including MPO, into the phagosome. The latter process creates a space between the microbe and the membrane of the phagosome, which contains a variety of granule components through which the secreted ROS would need to travel to reach the ingested microbe. Some ROS, e.g., ·OH, are highly reactive and would be expected to be scavenged quickly prior to reaching the microbe. Other ROS, e.g., H2O2, are less reactive and thus have longer diffusion distances. That H2O2 can reach the microbe and react with it is suggested by the OxyR-mediated transcriptional response in E. coli, which is elicited by reagent H2O2 and when complement-opsonized E. coli are ingested by intact phagocytes. This suggests that H2O2 formed in the phagosome can reach the microbe at concentrations adequate to initiate an OxyR response [218
]. MPO, being present in human neutrophils at concentrations no less than 1–2% of the dry weight of the cells [6
], would be expected to be present in the phagosome in very high concentrations. Being a highly cationic protein with an isoelectric point greater than 10 [6
], it can bind to the negatively charged surface of the microorganism and react there with H2O2 to initiate MPO-dependent oxidant formation in close proximity to the ingested microbe.
When iodide is the halide, iodination is detected [69
]. The fixed iodide can be localized in part in the phagosome by autoradiographic techniques [49
] and by the detection of radioiodide in the isolated phagosome [219
]. The bulk of the iodination appears to involve other neutrophil constituents [49
, 220
, 221
] and extracellular proteins [222
, 223
]; however, autoradiographic studies demonstrated the presence of silver grains lining the surface of the ingested organism [49
], indicating bacterial iodination as well. Chlorination and bromination also occur. Thus, free 3-chlorotyrosine and 3-bromotyrosine levels, used as markers of the reaction of MPO with H2O2 and chloride or bromide, rise in the peritoneal fluid of wild-type mice with experimentally induced sepsis but not (or to a considerably lesser degree) in septic mice deficient in MPO [207
]. The bromination observed in the absence of MPO may be a result of the presence of eosinophil peroxidase [224
]. 3-Chlorotyrosine has also been detected in tracheal aspirates from preterm infants, particularly those with respiratory distress [225
], in human atherosclerotic lesions [226
], in sputum specimens of patients with cystic fibrosis [227
], and in bronchoalveolar lavage fluid proteins of patients with acute respiratory distress syndrome, the latter in association with increased nitrotyrosine levels and MPO [228
]. Fluorescein-conjugated beads react with HOCl to form mono- and dichlorofluorescein [216
]. When human neutrophils were exposed to opsonized fluorescein-conjugated beads, 20 beads per cell became cell-associated, of which approximately two thirds were incorporated into sealed phagosomes. Near stoichiometric chlorination of the phagocytosed beads occurred [216
]. Of particular interest is the demonstration that bacteria sequestered in the phagosome are chlorinated by a MPO-dependent mechanism, as indicated by the presence of 3-chlorotyrosine and 3,5-dichlorotyrosine in bacterial proteins [229
, 230
]. In contrast, neutrophils failed to nitrate bacterial proteins in the phagosome under conditions in which chlorination was readily observed [230
]. Although it seems clear that HOCl is formed in adequate amounts to kill the ingested microorganisms, it would be expected to be scavenged to some degree if formed some distance from the microbe, as it travels through a fluid rich in scavengers, thus decreasing the amount of HOCl available for reaction with the ingested bacteria [229
]. However, chlorination of tyrosine residues of bacterial proteins does occur [229
, 230
], and this may be only the tip of the iceberg, as kinetically more favored (and likely more toxic) reactions of HOCl with, among others, sulfhydryl groups, iron-sulfur centers, and sulfur-ether groups would be expected to precede chlorination. Thus, although not all of the HOCl formed by leukocytes is used to attack the ingested organism, some of it is, which raises the question: how much is enough? The microbicidal power of HOCl is compatible with the need for only a portion of the HOCl formed.
H2O2 generation by neutrophils is required for optimum microbicidal activity
Enzymes that scavenge H2O2, such as catalase, protect certain organisms, e.g., coagulase-positive staphylococci, from the toxic effect of reagent H2O2 [231
, 232
] and neutrophils [233
] in vitro, and staphylococcal strains rich in catalase are more virulent in vivo [233
]. Furthermore, neutrophils from patients with CGD lack a respiratory burst, and the microbicidal defect in these cells can be reversed in part by the introduction of H2O2 into the cell [234
235
236
237
]. Glucose oxidase, which forms H2O2 without an apparent O intermediate, can be used for this purpose, indicating that H2O2 is effective even when the cells remain deficient in O. Finally, certain microorganisms, e.g., strains of streptococci, pneumococci, and lactobacilli, generate H2O2, and these organisms are killed well by CGD leukocytes [238
239
240
] and are rarely found in the lesions of infected patients. Presumably, they provide the H2O2 required for their own destruction by leukocytes that lack a cellular H2O2-generating system. This is supported by the finding that mutant strains of Streptococcus faecalis [241
] and Streptococcus pneumoniae [242
, 243
] with diminished H2O2 production are killed less well by CGD leukocytes than are the wild-type strains. These organisms also lack a heme catalase; however, the studies with mutant strains suggest that the level of H2O2 production may be more important than the catalase content in their susceptibility to killing by CGD leukocytes.
MPO is required for optimum microbicidal activity
Peroxidase inhibitors such as azide [244
, 245
], cyanide [244
, 245
], and sulfonamides [246
] decrease the microbicidal activity of normal neutrophils and have little or no effect on the microbicidal activity of MPO-deficient neutrophils, suggesting that they exert their effect on normal neutrophils largely by the inhibition of MPO. Neutrophil cytoplasts (neutroplasts) lack nuclei and are greatly depleted of cytoplasmic granules (and thus MPO) but have an intact respiratory burst [247
, 248
]. Neutroplasts phagocytose but do not kill Staphylococcus aureus unless the bacteria are coated with MPO [249
], suggesting that the respiratory burst is not sufficient for the killing of the organisms but that the additional presence of MPO is required. Similarly, stimulated neutroplasts do not lyse erythrocytes [250
] or inactivate 1-proteinase inhibitor [251
] unless MPO is added. Finally, human neutrophils, which lack MPO, have a microbicidal defect in vitro, although the defect is not as severe as in CGD. Thus, in 1969, Lehrer, Cline, and Hanifin [252
, 253
] described a diabetic patient with MPO deficiency and systemic candidiasis whose neutrophils had a prolonged candidacidal defect in vitro, whereas their staphylocidal activity was characterized by a lag period following which the organisms were killed. At this time, MPO deficiency was considered to be a rare event. However, the introduction of automated white blood cell counting techniques, which used the peroxidase stain, indicated that hereditary MPO deficiency was not uncommon in Europe and America (one in 2000–4000 [254
, 255
]), although it was less common in Japan (complete deficiency, one in 57,135 [256
]). Although some of these patients had clinical infections, most were well despite the demonstration of a neutrophil microbicidal defect in vitro [254
, 255
, 257
, 258
]. In one study comparing 100 patients with total or subtotal MPO deficiency to 118 with normal MPO levels, there was a statistically significant, higher incidence of severe infection and chronic inflammatory processes in the deficient patients [259
].
Does the microbicidal activity of human MPO-deficient leukocytes accurately reflect the contribution of MPO to the microbicidal activity of normal cells? Evidence has been provided suggesting that it may not [244 ]. Figure 5 demonstrates the effect of the peroxidase inhibitor azide on the microbicidal activity of normal and MPO-deficient neutrophils on three organisms, Lactobacillus acidophilus, coagulase-negative staphylococci, and Candida tropicalis. As reported earlier [252 , 253 ], the organisms were killed less well by MPO-deficient than by normal neutrophils. Azide markedly decreased the microbicidal activity of normal neutrophils and had no effect on the microbicidal activity of MPO-deficient neutrophils, suggesting that it exerts its effect on normal cells by the inhibition of MPO. Of particular interest is the observation that the microbicidal activity of MPO-deficient leukocytes is greater than that of azide-treated, normal cells [244 ]. This suggests that the contribution of MPO to the microbicidal activity of normal neutrophils may be greater than that suggested by studies with MPO-deficient neutrophils, as the latter cells appear to have adapted to the long-term absence of MPO with an increase in the activity of the MPO-independent (azide-insensitive) antimicrobial systems. Thus, the microbicidal activity of MPO-deficient cells appears to underestimate the contribution of MPO to the killing by normal cells.
|
It can be concluded from these studies that MPO is involved in the microbicidal activity of normal neutrophils, particularly in the early post-phagocytic period or when the microbial challenge is high, but that MPO-independent antimicrobial systems develop more slowly but are ultimately effective in MPO-deficient leukocytes, particularly when the microbial challenge is low. It should be emphasized that organisms differ in their susceptibility to oxygen-dependent antimicrobial systems. Thus E. coli are killed well by CGD and MPO-deficient leukocytes [264 ], fungi are particularly dependent on MPO for killing [252 ], and staphylococci are intermediate [253 ]. However, even if the MPO system is not required for the killing of a particular organism, it may normally do so in the early post-phagocytic period as a result of its rapid mobilization and power.
|
MPO-INDEPENDENT ANTIMICOBIAL SYSTEMS |
|---|
ROS operating in the absence of MPO
The staphylocidal activity of MPO-deficient neutrophils is inhibited by anaerobiosis, indicating that the microbicidal activity is at least in part dependent on oxygen ([222
]; see p. 437 in ref. [265
]). The respiratory burst of murine [260
] and human [222
, 266
267
268
269
270
271
272
273
274
] MPO-deficient leukocytes has been shown in a number of studies to be greater than normal, which may be a result of at least two mechanisms. First, MPO may be required for the termination of the respiratory burst [275
], and thus, its absence would lead to increased production of toxic oxygen metabolites. Second, as H2O2 is degraded in part by the MPO system in neutrophils, the absence of MPO would be expected to lead to a build-up of H2O2 as well as that of other oxidants dependent on H2O2 for their formation. These oxidants may eventually kill or at least contribute to the killing of the ingested organisms in the absence of MPO.
O and H2O2 are recognized products of the respiratory burst of phagocytes, which do not require MPO for their formation. O could theoretically be directly toxic to ingested organisms. However, many biologically important compounds react rather sluggishly with O, leading to the suggestion that O does not have the necessary reactivity to be directly toxic to ingested organisms. However, following are a few words of caution. The chemical reactivity of O is increased considerably in a nonpolar environment, as exists in the hydrophobic region of a membrane, where the reactions of O are not in competition with the proton-requiring dismutation reaction. Under these conditions, O is a powerful base with considerable nucleophilicity and reducing activity. Further, the protonated form HO2· is a considerably stronger oxidant than is O, raising the possibility that a local fall in pH, as might occur at a membrane surface or within a phagosome, may cause a shift in the HO2· 
O equilibrium toward the more potent, protonated form, with localized damage to a membrane or ingested organism. Further, the low, steady-state concentration of O would limit its dissipation by spontaneous dismutation, and this together with its relatively low reactivity allow it to diffuse over significant distances as through the ion channels of some cell membranes [276
], where it may be toxic at a distance through the formation of more reactive oxidants. H2O2 alone has antimicrobial properties at concentrations higher than those needed to generate toxic amounts of HOCl by the MPO system and thus, may contribute to the microbicidal activity of MPO-deficient phagocytes.
Reactive nitrogen intermediates (RNI)
Nitric oxide (NO·) is a recognized product of a cytokine-inducible NO· synthase in murine phagocytes where it contributes to microbicidal activity [277
]. It acts synergistically with ROS under some conditions [278
]. The production of NO· by human phagocytes, however, has been more difficult to demonstrate. Thus, in early studies, NO· formation by human phagocytes could not be detected under conditions in which NO· formation by murine phagocytes was readily apparent [279
280
281
]. However, human neutrophils appropriately stimulated have been shown to contain an inducible NO· synthase (iNOS) [282
283
284
], as do tissue macrophages from infected humans [285
286
287
288
]. It has been concluded that although human mononuclear phagocytes can produce iNOS when appropriately stimulated, NO· production by these cells is very low as compared with mouse phagocytes [289
, 290
].
NO· reacts with O to form peroxynitrite (ONO2–) [291
292
293
] (Fig. 6
), which can oxidize nonprotein and protein sulfdryl groups [294
]. ONO2–, at acid pH, is protonated to form peroxynitrous acid (ONO2H), which decomposes to form
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ABSTRACT
INTRODUCTION
