Pediatr. Res. 17: 662-664 (1983) Activation and Activity of the Superoxide- Generating System of Neutrophils from Human Infants RONALD G. STRAUSS(*~'AND ESTHER L. SNYDER Department of Pediatrics, University of Iowa College of Medicine, Iowa City, Iowa, USA Summary stimuli triggers activation of a reduced pyridine nucleotide oxidase that transfers single electrons from NADPH to molecular oxygen, We investigated the superoxide-generating system of neutro- thus reducing oxygen molecules to form superoxide anion (4). As phils obtained from human neonates and their mothers. Using a a consequence of activation, neutrophils experience an oxidative standard assay to measure the cumulative production of superox- burst characterized by increased oxygen consumption, by the ide anion over 15 min, neonatal neutrophils generated equivalent production of superoxide anion, hydrogen peroxide, hydroxyl or increased quantities of superoxide when compared to mothers radical, singlet oxygen, and chemiluminescence, and by increased and controls. Nanamoles of superoxide produced by 2.5 X lo6 activity of the hexose monophosphate shunt (4). The importance unstimulated neutrophils was 7.95 +. 3.45 for neonates, 2.25 f 1.50 of these oxidative reactions has been established by studies of for mothers, and 2.70 + 0.90 for controls. Comparable values for patients with chronic granulomatous disease. Their neutrophils 2 neutrophils stimulated by phorbol myristate acetate were 97.95 cannot mount an oxidative burst and, consequently, are unable to 2.10, 84.75 7.50, and 88.50 2.55, respectively (neonates > + + efficiently kill bacteria. controls, P 0.02). When studied by a continuous assay to assess < To more completely investigate oxidative metabolism in neo- kinetics of superoxide generation, neonatal neutrophils were more natal neutrophils, we studied superoxide generation for two rea- rapidly activated (P 0.02) than were cells from mothers or < sons. (I) Superoxide production is the key reaction in the initiation controls. Seconds elapsed from the addition of 0.667 pg/ml phorbol of the oxidative burst (1 1); accordingly, its characteristics must be until maximal rates of superoxide production were 39.3 2 2.88, defined. (2) Techniques are available to precisely measure the 49.1 3.12, and 52.2 2 4.32 for neonates, mothers, and controls, + kinetics (both activation and activity) of the superoxide-generating respectively. After complete activation, the rates of superoxide system (8, 9). Kinetic abnornalities of the oxidative burst have production were similar for all subjects (about 8 nmole/min/2 x been detected in neonatal neutrophils by chemiluminescence (15, neutrophils under a variety of conditions). lo6 17, 21), and further studies are warranted because of the impre- The increased activity of some aspects of oxidative metabolism cision of the chemiluminescence assay (2, 14, 15, 21). that occurs in neutrophils from human neonates might be ex- plained, at least in part, by a more easily activated superoxide- MATERIALS AND METHODS generating system that promptly initiates the oxidative burst. In contrast, those aspects reported to be decreased cannot be ex- Studies were approved by the local committee governing human plained by abnormalities of superoxide generation per se because investigations, and informed, written consent was obtained. Ve- maximal rates of superoxide production were comparable in neo- nous blood was collected from full-term infants (4-5 ml) and their natal and control neutrophils. Although the mechanisms respon- mothers within 24 h of birth. Adults served as controls. All subjects sible for decreased oxidative metabolism in neonatal neutrophils were healthy and were taking no medications on a regular basis. remain undefined, they apparently affect the oxidative burst at Cell suspensions containing greater than 90% neutrophils were steps subsequent to the early steps in superoxide generation. obtained by dextran sedimentation, Ficoll-Hypaque centrifuga- tion and hypotonic lysis by an isolation technique previously Abbreviation shown to yield uniform suspensions of morphologically mature neutrophils from infants, mothers, and controls (18). Neutrophils PMA, phorbol myristate acetate were suspended in Krebs-Ringer .phosphate . buffer (pH 7.4). Standard assay to measure the cumulative generation of super- Newborn infants are more susceptible to infections and expe- oxide. The ability of neutrophils to produce and release superoxide rience greater morbidity and mortality from infections when com- anion is usually assessed by measuring the total quantity of pared to older individuals. Although several abnormalities of superoxide dismutase inhibited ferricytochrome C reduction oc- neonatal body defense mechanisms have been described, reports curring over a defined period of time (19). The total volume of all are contradictory and controversy exists regarding the significance reaction mixtures was 1.6 ml, and all reactions contained 0.1 ml of each defect. Neutrophils provide an important defense against of 1.2 mM ferricytochrome C (80 pM final concentration) and 0.3 bacterial infections. Although complete agreement does not exist ml of a solution containing either superoxide dismutase or bovine regarding the properties of neonatal neutrophils, abnormalities of serum albumin (30 pg of protein final concentration). Stimulation chemotaxis, phagocytosis, microbicidal activity, and biochemistry reactions received 1.0 ml of neutrophil suspension (2.5 x lo6 cells) have been reported (Reviewed in 16). Studies of oxidative metab- plus either 0.1 ml opsonized zymosan (50 zymosan particles per olism are particularly perplexing. Increased activity has been neutrophil) or 0.1 ml PMA. PMA (Consolidated Midland Corp., reported for some aspects including oxygen consumption (lo), Brewster, NY) was initially dissolved in dimethylsulfoxide at a superoxide anion generation (1, 12), hydrogen peroxide produc- concentration of 1 mg/ml and frozen in aliquots. It was thawed tion (20), hexose monophosphate shunt activity (3, 10, 17), and and prepared fresh daily by dilution with phosphate-buffered nitroblue tetrazolium dye reduction (5, 6, 10). In contrast, de- isotonic saline (pH 7.3) so that the final concentration was 0.667 creased activity has been reported for chemiluminescence (7, 15, pg/ml. Unstimulated reactions received an identical quantity of 17, 21) and hydroxyl radical formation (1). neutrophil suspension plus 0.1 ml of buffer. Components were Activation of neutrophils by phagocytosis or exposure to soluble incubated with constant agitation at 37OC for 15 min. Reactions SUPEROXIDE-GENERATING SYSTEM 663 were stopped by plunging the vials into an ice bath, and neutro- linear portion of the curve intercepted the stable preactivation phils were immediately removed by centrifugation. Supernatant baseline. Activity of the superoxide-generating system was deter- fluids were transferred to clean tubes and promptly read spectro- mined by the linear rate of absorbance change that followed photometrically at 550 nm. The quantity of superoxide anion complete activation. The initial rate was consistently linear, and produced was expressed as nmol ferricytochrome C reduced in it was computed using measurements that encompassed the first 15 rnin by 2.5 x 10%eutrophils using the extinction coefficient 2 min. The change in absorbance then slowed, and the later rate E550 nm = 2.1 x lo4 m-' cm-' . of ferricytochrome C reduction was measured during the fifth Continuous production of superoxide anion. Both activation and minute after addition of PMA. As in the standard assay, super- activity of thesuperoxide-generating system were measured by the oxide anion generation was expressed as nmole of ferricytochrorne continuous measurement of suveroxide dismutase inhibited ferri- C reduced. cytochrome C reduction. A keviously described spectrophoto- Statistical analysis was performed in consultation with Dr. Leon metric assay (8, 9) was modified. The reaction was monitored at F. Burmeister, Department of Preventive Medicine and Environ- 37°C by Gilford model 250 spectrophotometer attached to a mental Health. The significance of differences between means was model 6050 chart recorder (Gilford Instrument Laboratories, tested by the two group t, paired t and sign tests. Oberlin, OH) with the following settings: wave length 550 nm; slit width 0.03 mm; calibrated absorbance 0.6; chart speed 1 inch/ RESULTS min. Cuvettes were filled with 1.1 ml of buffer, and after prewarm- ing at 37"C, 0.2 ml of neutrophil suspension (2 x 10%ells), 0.1 The total quantity of superoxide anion released and accumu- ml ferricytochrorne C (8 x lop5M final concentration) and 0.1 of lated in the reaction mixture as reduced ferricytochrorne C during either superoxide dismutase or buffer was added. Factors known 15 min of incubation (standard assay) is recorded in Table 1. to influence results of the assay such as pH, concentration of Maternal and control values were similar under all experimental divalent cations, and quantities of neutrophils and stimulants were conditions. The mean value for superoxide generation by unstim- carefully controlled (8). Temperature was precisely maintained at ulated neonatal neutrophils was nearly 3-fold greater than that of 37"C, and the reaction was allowed to stabilize (no further change controls; however, the difference was not statistically significant. in absorbance) for 2-5 min. Neutrophils were stimulated by Superoxide generation was markedly increased in neutrophils adding