Production of oxygen-centered radicals by neutrophils and macrophages as studied by electron spin resonance (ESR).

Neutrophils and macrophages undergo a respiratory burst and an increase in the activity of the hexose monophosphate pathway in response to particulate or soluble agents. The increase in oxygen consumption was found to be associated with the production of oxygen-centered radicals. The ESR technique of spin trapping showed that besides a superoxide spin adduct, a hydroxyl spin adduct is also produced. ESR is considered to be the least ambiguous technique for the detection of free radicals. The spin-trapping agents used for oxygen-centered radical detection are usually nitrones. The most commonly used nitrone is 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), which reacts with O2-. to form 5,5-dimethyl-2-hydroperoxypyrroline-N-oxide (DMPO-OOH) and with OH. to form 5,5-dimethyl-2-hydroxypyrroline-N-oxide (DMPO-OH). Although spin-adduct formation is considered to be the most direct technique for the detection of free radicals, some disadvantages are encountered. There has been considerable interest in the isolation of the O2-. generating activity from phagocytic cells. The enzyme can be extracted with deoxycholate and gel filtration indicates that it is a high molecular weight complex. Maximum activity was between pH 7.0 and pH 7.5. The Km value was 15.8 microM for NADPH and 434 micron for NADH, indicating that NADPH is the preferred substrate.


Introduction
Neutrophils undergo a respiratory burst in response to particulate or soluble agents, which results in the production of oxygen-centered radicals. The respiratory burst is not blocked by cyanide (1) and is associated with increased activity of the hexose monophosphate pathway (2). Although Iyer et al. (3) had shown that dioxygen was reduced to H202 during the respiratory burst of phagocytosing leukocytes, the linkage between the increase in oxygen consumption and production of oxygen-centered radicals was provided by the seminal discovery of Babior et al. (4) that neutrophils formed superoxide (02-) when stimulated with latex particles.
The Superoxide-Generating System An enzymatic basis for the oxidation of pyridine nucleotides in activated neutrophils was indicated by Patriarca et al. (13). The 021-generating enzyme resides in the plasma membrane (14). Cohen et al. (15) found the enzyme in phagocytic vacuole walls. The enzyme activity is not expressed in membrane fractions from unstimulated neutrophils from normal subjects and from stimulated neutrophils from patients with chronic granulomatous disease (CGD) (16).
There has been considerable interest in the isolation of the 02--generating activity from various phagocytic cells (17)(18)(19)(20)(21)(22)(23)(24)(25). Very little progress has been achieved until recently because of the use of procedures giving an extremely unstable enzyme. A procedure for the extraction and isolation of a highly active and stable enzyme from stimulated guinea pig neutrophils was reported by Bellavite et al. (24). The enzyme was extracted with deoxycholate and gel filtration indicated that it is a high molecule weight complex. Maximum activity was between pH 7.0 and pH 7.5. The Km value was 15.8 ,uM for NADPH and 434 ,uM for NADH, indicating that NADPH is the preferred substrate.
A number of alternative enzyme activities have also been proposed as being responsible for the 02--generating activity. These have included myeloperoxidase (MPO), D-amino acid oxidase, NADH oxidase, and cytochrome b. MPO is able to catalyse the oxidation of reduced pyridine nucleotides at low pH and in the presence of Mn2 (26). However, a normal respiratory burst and NADPH oxidase activity are seen in neutrophils from MPO-deficient patients (27,28). A similar argument rules out D-amino acid oxidase. This enzyme activity is normally present in neutrophils from CGD patients which have no G2--generating activity (29). The production of G2 and H202 by an NADH oxidase in guinea pig neutrophils has been described by Badwey and Karnovsky (30). However, a rather modest decrease in the activity of the enzyme in neutrophils from CGD patients was observed in contrast to the total absence of NADPH oxidase activity. The arguments presented so far clearly indicate that NADPH oxidase is the enzyme responsible for the production of G2by stimulated neutrophils. There is also considerable interest in the structural components, cofactors and mechanism of action of the enzyme. The enzyme has been characterized as a flavor protein (19,(23)(24)(25). However, this has recently been disputed because various extensively purified preparations of the enzyme have been found to contain varying ratios of flavin to enzyme activity and recent preparations have been found to contain little flavin (31). A b-type cytochrome associated with the phagocytic vacuole wall has been implicated in the NADPH oxidase activity (32). A proposal that the cytochrome b may be the oxidase itself (33) is poorly supported because the cytochrome appears to be absent only in some CGD patients (34,35). It can be noted that cytochrome b autoxidation has been ruled out as a source of G2" generation in mitochondria (36). The possibility that a b-type cytochrome forms part of the NADPH oxidase complex is, however, not excluded since the cytochrome copurifies with the enzyme activity and a constant ratio is maintained throughout the purification (25). Other components which have been claimed to be associated with the enzyme activity are calmodulin (37,38) and quinones (39). However, the exact involvement of all these components in the oxidase activity remains to be elucidated.
Macrophages behave metabolically in much the same way as neutrophils in response to phagocytic and a variety of surface stimuli, and the mechanism of G2production by these phagocytic cells is basically the same as in neutrophils (40,41).
Reduction of ferricytochrome c, controlled by inhibition of the reaction by superoxide dismutase, as originally employed by Babior et al. (4) is commonly used to assay the production of G2by phagocytic cells. The reaction has the advantage that it can be followed continuously by optical spectroscopy (42). It should be borne in mind that regurgitation of MPO by phagocytosing neutrophils (43) may result in the reoxidation of ferricytochrome c, in the presence of H202 (44). Hydroxyl radicals (OH), formed as a consequence of G2production, may also reduce ferricytochrome c by a mechanism whereby a radical formed after hydrogen is abstracted from the outer surface of the protein reduces the protein by electron tunnelling (45). Inhibition by superoxide dismutase alone would not distinguish this component of ferricytochrome c reduction.
Electron spin resonance (ESR) is considered to be the least ambiguous technique for the detection of free radicals. However, even when concentrations of G2exceed those normally required for detection (10-8 M) no ESR spectrum is observed in aqueous solution under physiological conditions. This is because of the very short relaxation time of the G2radical. However, the technique of spin trapping allows the formation of stable free radical products thereby permitting their detec-   (46). The most commonly used nitrone is 5,5dimethyl-1-pyrroline-N-oxide (DMPO) which reacts with O2to form 5,5-dimethyl-2-hydroperoxypyrroline-N-oxide (DMPO-OOH) and with OH to form 5,5-demethyl-2-hydroxypyrolline-N-oxide (DMPO-OH). A typical spectrum is given in Figure 1. The g factors, hyperfine components, and splitting constants for the spin adducts of DMPO and other nitrones are given in Table 1. Green et al. (47) were the first to use the spin trap DMPO to detect O2production by stimulated neutrophils. This work also represents the first use of spin trapping with intact cells. The ESR spectrum of neutrophils stimulated with phorbol myristate acetate (PMA) was consistent with that of a mixture of the products of the reaction of DMPO with OH and O2.-In neutrophils stimulated with IgG-coated latex particles, the ESR spectrum was that expected from the spin adduct DMPO-OH. In this system a contribution of the spin adduct DMPO-OOH was seen in the presence of azide. No spin adduct formation was observed in the presence of superoxide dismutase. However, the spectrum of the DMPO-OH was still observed, with reduction in intensity, in the presence of catalase.
Green et al. (47) and Rosen and Klebanoff (48) were able to detect only the DMPO-OH spin adduct with neutrophils stimulated with opsonized zymosan. The signal could be virtually abolished by superoxide dismutase, modestly decreased by catalase, and appreciably decreased by mannitol. Neutrophils from a patient with CGD did not show the DMPO-OH signal, while neutrophils from a patient with hereditary MPO deficiency showed an enhanced signal with 25% increase in intensity. The signal was reduced to the intensity level seen with normal neutrophils by addition of purified MPO to the incubation mixture (48). It was strongly suppressed by superoxide dismutase or mannitol but not by catalase.
It has been suggested that the work of Green et al. (47) and Rosen It may be noted that DMPO can best compete with spontaneous dismutation of O2at high pH because of low pH the advantage of the higher rate constant of reaction (8) relative to reaction (7) is cancelled by the increased rate of dismutation of O2- (49). The rate constants shown for reactions (7) to (9)  SOD depressed both DMPO-OOH and DMPO-OH spin adduct signals while catalase did not totally abolish the signal. This argues in favor of decomposition of DMPO-OOH to DMPO-OH; however, no such effect was observed with spin trapping of 02-from isolated guinea pig neutrophil NADPH oxidase (38) and macrophages stimulated with PMA (71). Hydroxyl radical formation by phagocytosing neutrophils and macrophages is presumed to proceed by the Fenton reaction, Fe2" + H202 -* Fe3`+ OH' + OH- (10) and redox cycling of the metal by O2-, Fe3 + O -* Fe2 + 02 (11) in phagocytosing neutrophils. The sum of reactions (10) and (11) is°2 0 + H202 . 02 + OH + OH- (12) Reaction (12) was called the Haber-Weiss reaction by Beauchamp and Fridovich (53), since it occurs in the reaction scheme of Haber and Weiss (54) to explain the catalytic decompostion of H202 by iron salts. This name is now sanctioned by usage. There are many difficulties concerning this reaction, not the least being the manner in which it occurs. The reaction is thermodynamically feasible (55) but kinetically hindered having a negligible rate constant (56). Metal catalysis of the reaction has been demonstrated with Fe-EDTA (57,58), the estimated rate constant of the catalyzed reaction being approximately 1.0 x 103 M -1sec-1 (57). Of biological interest is the occurrence of the reaction with iron chelates of ADP (59) or ATP (60) and picolinic acid (61), with nonprotein-bound iron of body fluids (62,63), and with the iron associated with transferrin (57,64,65), lactoferrin (66)(67)(68), and ferritin (69). The occurrence of the Haber-Weiss reaction in the presence of lactoferrin (Fig. 2) has a direct bearing in the production of OH radicals by phagocytosing neutrophils since these cells release lactoferrin into the phagolysosomes and extracellularly during degranulation (70). Ambruso and Johnston (66) showed that iron-saturated lactoferrin enhanced the production of OH radicals by human neutrophils stimulated with opsonized zymosan or PMA and by a particulate fraction prepared from the neutrophil homogenates. Production of 02by purified NADPH oxidase obtained from guinea pig neutrophils (24), stimulated with PMA, has been demonstrated by spin trapping of the radical with DMPO. The signal observed was mainly that of DMPO-OOH (38).
In the presence of iron-saturated transferrin (64), the intensity of the signal was reduced by about 40% indicating that the transferrin iron (Tr-Fe + was competing with DMPO for free O2radicals, Tr-Fe3+ + 0--) Tr-Fe2+ + 02 (13) production of OH radicals resulted when H202 was added to the system, Tr-Fe2" + H202 --Tr-Fe3+ + OH + OH- (14) The signal of the DMPO-OH spin adduct was resolved in the ESR spectrum by computer stimulation which gave g = 2.0050, aN = aH = 14.9G (Fig. 3). The Haber-Weiss reaction [reaction (12)], however catalyzed, should be inhibited by either superoxide dismutase or catalase. A large inhibition by catalase (-50%) of the intensity of the DMPO-OH spin adduct signal by human neutrophils stimulated with opsonized zymosan (48) and by Bacille Calmette-Guerin (BCG)elicited mouse peritoneal macrophages activated with PMA (71). The intensity of the DMPO-OH spin adduct signal can be suppressed by about 70% by the OH' radical scavengers mannitol (48), dimethyl sulfoxide (71) and the Fe + scavenger desferrioxamine (71) in stimulated neutrophils and macrophages. It is not clear why catalase does not totally abolish OH' radical production.
Catalase is however known to be ineffective as a scav- enger at low concentrations of H202. The use of OH radical scavengers still leaves 30% of the DMPO-OH spin adduct signal unaccounted for. Evidence for production of carbon-centered radicals in activated macrophages has been obtained by means of the lipophilic spin trap 5-octadecyl-5,3,3-trimethyl-1-pyrroline-N-oxide (71). However no clear relationship has been established between these radicals and the unaccounted DMPO-OH spin adduct signal. An alternative mechanism to the iron-catalyzed Haber-Weiss reaction has been proposed to explain OH' radical production by phagocytosing neutrophils. These cells release MPO from the azurophil granules during degranulation (43). This results in catalysis of the reaction: MPO H202 + Cl -HOCI + OH- (15) HOCI can react with O2 , possibly producing OH (72).