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Table 7-3 Major ROS and Their Metabolism

Oxidative Burst and Oxidant Metabolites

The generation of toxic oxygen metabolites and ROS is the cardinal characteristic of the neutrophil,

though is also a defensive quality of the monocyte/macrophage. Their essential antimicrobial properties

are equally destructive to host tissues and implicated in the pathophysiology of many inflammatory

disorders (Table 7-3). Thus, a delicate balance must be maintained between ensuring elimination of the

offending pathogen and minimizing host damage.

A free radical is any species possessing one or more unpaired electrons. They are categorized under

the broader term ROS, which encompasses all molecules capable of radical formation. Despite a very

brief existence (10−11 to 10−6 seconds) extensive damage may occur through the induction of free

radical chain reactions. Species underlying both physiologic and pathophysiologic inflammation include

the following: the superoxide anion (O2

•−), the hydroxyl radical (•OH), hydrogen peroxide (H2O2

), the

singlet oxygen, and the reactive nitrogen intermediates nitric oxide (NO) and peroxynitrite (ONOO−)

(Table 7-3). Counterintuitive to their role in cell destruction, recent evidence also supports a role of

ROS in intracellular signal transduction.32 Thus, in addition to their antimicrobial properties, ROS can

modulate the immune response by activating inflammatory cells and inducing proinflammatory cytokine

secretion.33,34

2 Implicit with the capacity for pathogen elimination is the potential for destruction of host tissues.

Hence, numerous regulatory mechanisms provide temporal and spatial control of the ROS production.

The NADPH oxidase complex itself exists in a disassembled state, and only upon cell activation and the

need for ROS production are the subunits approximated and enzymatic function enabled.35 In addition,

high plasma and tissue concentrations of proteinase inhibitors provide continuous surveillance and

systemic control. However, such regulation is incomplete as evidenced by diseases such as rheumatoid

arthritis, chronic obstructive pulmonary disease, and autoimmune vasculitis, which are the consequence

of damage due to neutrophil-derived products.35

NADPH oxidase is a heteromeric complex composed of six subunits: flavocytochrome b558, the

electron transporting apparatus comprises gp91(phox) and p22(phox); the cytosolic complex p40(phox),

p47(phox) and p67(phox); and the oxidase factor rac-2 (Fig. 7-3).35 Microorganisms or high

concentrations of chemoattractants bind to cell surface receptors and initiate oxidase activation,

heralded by phosphorylation of p47(phox). Cytochrome b558 (gp91 and p22), which exists within the

plasmalemma and the membranes of specific granules and secretory vesicles, is recruited by

phagocytosis and granule fusion. Phosphorylation of p47(phox) induces a conformation change that

enables its incorporation within the membrane, wherein it facilitates the translocation of rac2 and

p67(phox), and stabilizes the association of this cytosolic complex with cytochrome b558, thereby

rendering the complex functional.35

At the redox center of the oxidase an electron is transferred from NADPH to oxygen, thereby

generating superoxide.36

O2 + e-=O2

Other mechanisms of superoxide production include uncoupling of xanthine dehydrogenase system,

uncoupling of mitochondrial and endoplasmic reticulum (ER) electron transport chains, and

nonenzymatic reactions such as autoxidation of hemoglobin.37,38

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Figure 7-3. NADPH oxidase assembly. In the resting neutrophil, the cytochrome subunits gp91 and p22 are tightly bound in the

membrane. P47(phox), p67(phox) and rac-s complex are in the cytosol. On activation, GDI releases rac-2, and p47(phox) becomes

phosphorylated. This causes translocation of rac-2, p47(phox), and p67(phox) to the membrane and complex formation with the

cytochrome components, thereby completing the assembly of the active oxidase. (Redrawn from Burg ND, Pillinger MH. The

neutrophil: function and regulation in innate and humoral immunity. Clin Immunol 2001;99(1):7–17.)

Superoxide is relatively weak and of low bactericidal potency. However, its membrane permeability

and role as a reactant in reactions yielding highly toxic products confers upon it a high potential for

cellular and tissue damage.36

Superoxide can spontaneously or enzymatically (superoxide dismutase) dismutate into hydrogen

peroxide.35,36

It can also be converted to the more potent hydroxyl radical through the metal-catalyzed Haber–

Weiss reaction.35,36

or through the Fenton equation35,36

Under physiologic conditions, lactoferrin found in neutrophil specific granules provides the iron

catalyst for the Haber–Weiss reaction. In the Fenton reaction, superoxide or other biologic reducing

agents such as lactate or ascorbate donate electrons to generate the ferrous ions required to react with

hydrogen peroxide to produce the hydroxyl radical.35,36,39

The hydroxyl anion is highly reactive and induces DNA strand breaks and base hydroxylations leading

to adenosine triphosphate (ATP) depletion and gene mutations. It can attack lipid side chains of

membrane phospholipids to form hydrogen peroxide and lipid hydroperoxides in a process called lipid

peroxidation. These products can disrupt membrane function, serve as substrates for the production of

cytotoxic aldehydes, or uncouple calcium-ATPase and increase cytosolic calcium concentration. Recent

data also support a mechanism by which oxidation of critical sulfhydryl residues on the ryanodine

receptors induces an “open” configuration and a leak of intracellular ER calcium into the cytosol.40 This

elevation of cytosolic calcium activates calcium-dependent proteases and phospholipases that propagate

cellular damage.39

Superoxide can react with nitric oxide to produce peroxynitrite (ONOO–) and hydroxyl radical.36,41

The hemoprotein MPO yields the potently bactericidal HOCl from the reactants chloride and H2O2

.

HOCl oxidizes amino acids, nucleotides, and hemoproteins, can activate neutrophil collagenases and

permit unabated elastase injury by inhibiting α1-antitrypsin, and contribute to hydroxyl radical and

singlet oxygen production.33,39,42 Though short-lived, subsequent reactions with secondary amines

generate secondary chloramines, which are equally toxic but much more stable. These metabolites can

oxidize similar cellular components. They can combine with halide anions to generate toxic free halides

or with taurine chloramines that induces membrane attack complex (MAC) complement

formation.33,39,42

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In light of the pivotal role of MPO during inflammation and pathogen elimination, it is surprising that

MPO deficiency is common and relatively benign. Though MPO-deficient neutrophils show early

depressed bacterial killing, bactericidal function normalizes within 60 minutes.43 It is hypothesized that

though bacterial killing is impaired, post-phagocytosis-oxidase–dependent neutrophil apoptosis is

normal, resulting in appropriate regulation of the inflammatory response.43

Singlet oxygen, a highly reactive and extremely short-lived species, is formed by an input of energy

to O2

that reverses the spin direction of one of the outermost unpaired electrons away from a parallel

spin. It is produced during reactions of the MPO–H2O2–halide system and is a potential product of

superoxide dismutation and the Haber–Weiss reaction. It is highly electrophilic, reacting with

compounds containing electron-rich double bonds and may react with membrane lipids to produce

peroxides.39

The destructive potential of these ROS for both host and pathogen necessitates a mechanism of

continuous tight spatial and temporal regulation. The oxidase complex itself exists spatially

disaggregated; only upon cellular activation are its constituents assembled and enzymatic function

restored (Fig. 7-3).35,43 As elegant is the mechanism by which to control ROS production, so too are the

measures employed to eliminate these products when no longer needed. Superoxide, the proximal

reactant necessary for many of the ROS generating reactions, is removed by both spontaneous and

enzymatic (superoxide dismutase) dismutation to H2O2

(Fig. 7-4). H2O2

is subsequently reduced to

oxygen and water by catalase.36,39 In the extracellular environment, this function is performed by GSH

peroxidase, a selenium-dependent enzyme that reduces H2O while oxidizing reduced GSH to its oxidized

form. The utilization of N-acetylcysteine, a reducing agent that restores GSH, reduces hepatocellular

injury in an animal model of warm liver ischemia/reperfusion.32 It has also been shown to reduce

contrast-induced nephropathy in patients undergoing imaging procedures requiring the use of iodinated

contrast.44 Mechanisms for preventing hydroxyl radical-induced tissue damage include the binding of

transition metal ions by albumin, ceruloplasmin, haptoglobin, lactoferrin, and transferrin.33,36,39 Taurine

is a scavenger for HOCl. Other antioxidants that may assist in controlling the reaction include vitamins

E (tocopherol) and C. Vitamin C has many antioxidant properties, including the ability to regenerate αtocopherol. It can prevent activation of neutrophil-derived collagenase and is a powerful scavenger of

HOCl, superoxide, singlet oxygen, and hydroxyl radicals. Carotenoids have long double bonds to attract

and sequester free radicals. Uric acid is a powerful scavenger of water-soluble radicals such as HOCl and

singlet oxygen. It can also bind copper and iron ions to suppress hydroxyl radical formation. Stress

proteins or heat-shock proteins (HSPs) are induced by oxygen radicals and ischemia and may play a role

in defense. Furthermore, heme oxygenase-1 (HO-1) catalyzes the cleavage of heme to biliverdin, which

is subsequently converted to bilirubin, an efficient free radical scavenger.33,36,39

All of the aforementioned participants of the NADPH oxidase are vital for health, as evidenced by

those who suffer from chronic granulomatous disease (CGD).35,43 These patients have deficient

superoxide production and experience ineffective inflammatory reactions to infection. They commonly

suffer from repeated bacterial infections (pneumonia, cutaneous abscesses and hepatic and perihepatic

abscess, and osteomyelitis) by organisms that are catalase positive (Staphylococcus aureus). Organisms

that produce large amounts of peroxide are less of a threat as the neutrophils can utilize bacterial

peroxide to produce toxic metabolites. The use of prophylactic antibiotics and IFNγ has reduced the

frequency of serious infections in this patient population.35,43

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Figure 7-4. Scavengers of ROS. (Redrawn from Klebanoff SJ. In: Gallin JI, Snyderman R, eds. Inflammation: Basic Principles and

Clinical Correlates. 3rd ed. Philadelphia, PA: Lippincott Williams & Wilkins; 1999:723.)

Neutrophil Extracellular Traps and NETosis

The arsenal of antimicrobial products and mediators possessed by neutrophils to fulfill the tasks of

killing and eliminating pathogens is vast and impressive. However, another distinct antimicrobial

“weapon” was recently described, in which neutrophils extrude a lattice of chromatin and histones to

entrap and then kill invading pathogens. The characteristic feature is the presence of neutrophil nuclear

DNA in the extracellular space. These structures were entitled neutrophil extracellular traps (NETs), and

the process of NET formation called NETosis. Attached to this DNA backbone is an array of microbicidal

agents that are typically compartmentalized inside the azurophilic, specific and gelatinase granules:

neutrophil elastase (NE), cathepsin G, and MPO. Distinct from apoptosis, the process does lead to cell

death, though clearly contributes to pathogen control. It is conserved in various vertebrates, insects, and

even plants, and several microorganisms exhibit mechanisms of resistance to NETs, both of which may

highlight the importance of this process. Recent data suggest that excessive NETosis or ineffective NET

clearance may underly diseases or syndrome characterized by excessive inflammation, including

autoimmunity.45–47

Pathogens (i.e., bacteria, fungi, viruses, and protozoa) and their subcellular components, such as LPS,

M1 protein, and fMLP induce NET formation. Similarly, sterile insults, inflammatory stimuli (e.g., PAF,

IL-8, TNFα, NO), and chemical compounds (PMA, ionomycin) all can stimulate NETosis. The process

begins with engagement of the aforementioned stimuli with TLRs, Fc receptors, cytokine, and

complement receptors located on the neutrophil cell surface. Subsequent calcium release stimulates PKC

activity and assembly of functional NADPH oxidase, leading to ROS and NO production. ROS is/are

requisite for NET formation, as antioxidant scavengers (e.g., N-acetylcysteine) attenuate the process.

Interestingly, individuals with CGD, a disease characterized by insufficient ROS production, inefficiently

produce NETs. The chromatin undergoes decondensation followed by mixing of nuclear and cytoplasmic

components with granulate antimicrobial contents. Plasma membrane rupture ensues with the release of

chromatin decorated with granular proteins into the extracellular space.45–47

NETs are able to trap almost all pathogens, including those too large to be phagocytosed. Indeed, it is

proposed that trapping by NETs is one of the most important functions that limits microbial spread. The

antimicrobial functions of NETs are mediated by the microbicidal compounds accompanying the

chromatin: NE, MPO, histones, cathepsin G, proteinase 3, lactoferrin, calprotectin, and antimicrobial

peptides such as defensive or the cationic LL37. However, the histones H1, H2A, H2B, H3 and H4

themselves also possess powerful antimicrobial effects.

As fundamental as NETosis is for microbial defense, excessive formation or ineffective clearance of

NETs is proposed to lead to several pathologic conditions. During acute lung injury a high concentration

of stimulating factors promotes NETosis and the release of NE, LL-37, and ROS, which may exacerbate

injury. In cystic fibrosis, the high concentrations of DNA due to NETosis are thought to underlie the high

mucus viscosity.45–47 Several autoimmune diseases are also characterized by perturbed NETosis, and

more than 70% of NET components are potent autoantigens. Small vessel vasculitis is characterized by

antineutrophil cytoplasmic antibodies reactive against PR3 and MPO. Externalization of IL-17 in the

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