Inhibition of human neutrophil oxidative metabolism and ...

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Basil O'Connor Starter Research Fund of the National Foundation of the March of Dimes, the. Leukemia Task Force, and Grants HLI,7132. HL18. H120695 ...

Inhibition of Human Neutrophil Oxidative Metabolism and Degranulation In Vitro by Nitroblue Tetrazolium and Vitamin E John E. Repine, MD, Gundu Rao, PhD, Gregory D. Beall, MD, and James G. White, MD

The effect of a mixture of nitroblue tetrazolium (NBT) and vitamin E on the metabolism and ultrastructure of polymorphonuclear leukocytes (PM-Nf) from normal subjects or patients with chronic granulomatous disease (CGD) was determined in vitro. Increasing concentrations of NBT and vitamin E progressively decreased rates of oxygen consumption and 1-"4C-glucose oxidation by normal PMIN stimulated with particulates to a degree that exceeded either agent alone. NBT-vitamin-E also inhibited vacuole formation and the cv-tochemical release of mveloperoxidase-positive granules. The depressed oxidative metabolism and degranulation of NBT-vitamin-E-treated control PMIN closely approximated the blunted responses of CGD PMN which were similar alone or in the presence of NBT-vitamin-E. In contrast to these effects, the highest concentration of NBT-vitamin-E used in the study did not damage. decrease rates of unstimulated oxidative metabolism of, or impair ingestion of particulates by control or CGD PMI;N. NBT and vitamin E impose a state on normal PMN which is remarkably similar to that observed in PMIN from patients with CGD. (Am J Pathol 90:659-674, 1978) METABOLIC AND MIORPHOLOGIC EVENTS that accompany phagocvtosis of bacteria by polvmorphonuclear leukocytes (PM1N) have been characterized in many investigations. Foreign microorganisms are incorporated in surface membrane invaginations w^hich seal their connections to the cell wall and migrate into the cy-toplasm as isolated vacuoles.l Azurophilic and specific granules fuse with phagocytic vacuoles and discharge a variety of enzymes around ingested bacteria.2 Engulfment and degranulation bv PMIN are paralleled by increased metabolic responses which include consumption of oxygen, oxidation of I-"C-glucose. and formation of hydrogen peroxide (H202), superoxide (02-, and hvdroxyl radical (OH-).34 The combined actions of granule-bound enzxmes and by-products from these metabolic reactions appear necessary for optimal PNMN bactericidal activity as evidenced by the abnormal microbicidal From the Pulmonary Section of the Department of Medicine and the Hematology Section of the Department of Pediatrics. University of Minnesota Health Sciences Center, Minneapolis. Minnesota Supported in part by the Minnesota Medical Foundation, the Minnesota Lung Association. the Basil O'Connor Starter Research Fund of the National Foundation of the March of Dimes, the Leukemia Task Force, and Grants HLI,7132. HL18. H120695, CA11996, GM-A M-22167. AM 15.317. and CA08832 Dr. Repine is an Established Investigator of the American Heart Association Dr Beall is a recipient of Public Health Service Research Fellowship IT32-HI07009-01 Al. Accepted for publication November 8. 1977. Address reprint requests to Dr John E Repine. Department of Mledicine, Box 13 Maso Memorial Building, University of Minnesota. Minneapolis, MN 55455 659

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activity of PMN deficient in granule constituents and/or oxidative metabolism .3,7-10 Considerable attention has been directed toward determining the trigger mechanism responsible for the burst of respiration in activated neutrophils. In particular, the exact role of the highly reactive unstable oxygen intermediates, especially superoxide anion, has been sought in an effort to explain normal PMN activation as well as the failure of PMN from patients with chronic granulomatous disease (CGD) to develop the postphagocytic respiratory burst.3.4,ll,U2 Measuring the conversion of nitroblue tetrazolium (NBT) to insoluble blue formazan has been the principle means for evaluating the redox activity of normal or abnormal PMN.13 NBT reduction is caused by activated control neutrophils but not by stimulated CGD PMN in which the respiratory burst is absent.'"15 Additional studies have indicated that the reduction of NBT by PMN following particle ingestion is due primarily to oxidase-dependent generation of superoxide anion.13 Although considerable work has been done to identify the mechanism by which NBT is reduced in activated PMN, little effort has been made to determine if NBT might inhibit neutrophil function. Recent investigations in this laboratory have shown that NBT inhibits the response of blood platelets to aggregating agents and that the inhibiting action of the dye is potentiated by vitamin E.16 The curious interaction of NBT and vitamin E suggested that the combination scavenged or blocked free radicals generated during platelet activation by aggregating agents, thereby inhibiting development of cell stickiness and prostaglandin synthesis. In the present investigation we have evaluated the influences of NBT and vitamin E on the activation of neutrophils during phagocytosis of particulates. Results of the study indicate that the combination of NBT and vitamin E markedly inhibits the respiratory burst and degranulation of PMN during uptake of bacteria without influencing the ability of the cells to engulf the organisms. Thus, NBT and vitamin E together impose a state on normal neutrophils remarkably similar to that observed in PMN from patients with CGD. Materials and Methods The present investigation was approved by the Human Volunteers Committee of the University of Minnesota. Blood was donated by 10 control subjects and 2 patients with CGD. All individuals were drug-free and in good health at the time of study. The diagnosis of CGD had been established in the affected patients by appropriate criteria. The patients had a history of frequent infections, and their PMN failed to reduce NBT dye and kill catalase-positive microorganisms normally in vitro.Ael?,v

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The methods used in this investigation have been reported in detail previously."11 Briefly, blood was obtained by venipuncture in syringes containing 40 units of heparin/ml and was allowed to sediment in a vertical position for 90 minutes. Leukocyte-rich plasma was transferred to plastic tubes and sedimented at 200g for 12 minutes. The platelet-rich supernatant was discarded and the leukocyte pellet was triply washed, counted, and resuspended in Hanks' balanced salt solution (HBSS) containing 8 units of heparin/mi."' Serum was obtained from 5 normal subjects, Type AB, by venipuncture, allowed to clot on ice, rimmed, recovered by centrifugation, pooled, and frozen at -70 C for not more than 2 weeks." Staphylococcus aureus organisms, 502A, periodically tested for strain identification, were heat-killed (HKB) following incubation for 30 minutes in a 70 C water bath." Nitroblue tetrazolium (NBT) (Sigma Chemical Co., St. Louis, Mo.) was suspended in HBSS and was then sonicated for 2 minutes prior to use." Vitamin E (Sigma Chemical Co.) (D-a-tocopherol) was similarly suspended and sonicated in 95% ethanol." OxNrgen consumption was measured on a Yellow Springs electrode monitor (Yellow Springs Instrument Co., Yellow Springs, Ohio) coupled to a Beckman recorder (Beckman Instruments, Inc., Fullerton, Calif.) with moving graph paper." Samples were maintained at 37 C by a circulating water bath and stirred magnetically. The slope of the recorder tracing was measured over a 15-minute period and converted to d1 02/hr/4 X 10' PMN. Rates of oxvgen utilization bv unstimulated PMN were determined for 3-ml samples containing 4 X 10' PMN/ml, 8% serum, and HBSS. Oxygen consumption was also measured for 3-ml mixtures containing 4 X 10' PMN/ml, HKB adjusted to vield a ratio of 50 KHB per PMN, 8% serum, and HBSS. Various concentrations of NBT and/or vitamin E were added to these mixtures prior to study. At the end of analysis, leukocyte samples were fixed in glutaraldehyde and osmic acid and embedded in Epon 812 (Shell Chemical Co., New York, NY) for study in the electron microscope.' Peroxidase activity was identified in paired samples of PMN at the ultrastructural level by a modification 21 of the method of Graham and Karnovsky.22 Thin sections of unreacted cells were stained with uranyl acetate and lead citrate before examination in a Phillips 301 electron microscope (Phillips Electronic Instrument, Mount Vernon, NY). Samples reacted for peroxidase were examined without section staining to enhance contrast. The oxidative metabolism of PMN was examined using isotopically labeled glucose as previously described."3 Briefly, "ICO2 produced by oxidation of 1-"4C-glucose was trapped on a sodium-hvdroxide-saturated filter paper suspended from the stopper of a 15-mi flask containing 4 X 10' PMN, 8% serum, HBSS, 50 HKB per PMN, NBT, and/or vitamin E. Parallel controls were run with various combinations of serum, HBSS, NBT, and/or vitamin E to determine oxygen consumption or glucose oxidation in the absence of PMN.

Results oc cc

The effect of increasing concentrations of NBT or vitamin E on the consumption of oxygen by mixtures of control PMN and HKB were determined in vitro (Table 1). In the absence of NBT or vitamin E, control PMN stimulated with 50 HKB:PMN consumed oxygen at a rate of 24 ± 1.8 M1/hr/4 X 106 PMN. When increasing concentrations of NBT or vitamin E were added to these PMN-HKB suspensions, oxygen consumption decreased in a dose-dependent manner. Incubation of HKB-treated PMN with various increasing combinations of NBT and vitamin E together progressively decreased oxygen consumption to a degree that

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Table 1-Consumption of Oxygen* by HKB-Stimulated PMN Treated With Various Concentrations of NBT and/or Vitamin E

Concentration of vitamin E (mg/ml)

Concentration of NBT (mg/ml) 0

0.08 0.17 0.25 0.33

0

4.2

8.3

12

17

25

24 15 9 6 5

18 13 10 6 5

12

10 5

10 8 5 5

4

4

10 6 4 5 3

7 5 4 3 3

11

In /l 02/hr/4 x 106 PMN

exceeded the effect of either agent alone. Final NBT-vitamin-E concentrations of 0.33 mg/ml and 25 mg/ml, respectivelv, consistently and profoundly inhibited oxygen consumption by control PNIN stimulated with bacteria (3.3 ± 0.9 Mi/hr/4 X 106 PMN). These concentrations were used in the remaining studies of control and CGD PMIN. Oxygen consumption bv unstimulated control PMN treated with 0.33 mg NBT/ml and 25 mg vitamin E/ml was statistically the same as that of control PMIN (Table 2). Following stimulation with 50 HKB per cell, normal PMIN treated with NBT-vitamin-E increased their rate of oxygen consumption only two-fold compared with a 15-fold increase observed for untreated control PMN. The resting rates of oxygen consumption by CGD PMN in the presence or absence of NBT-vitamin-E were comparable to control PNIN treated with NBT-vitamin-E. Addition of 50 HKB per cell did not increase rates of oxvgen consumption by untreated or treated CGD PNIN. Glucose Oxidation

The pattem of l-"4C-glucose oxidation by normal or CGD PMN treated with NBT and/or vitamin E wvas similar to that observed for oxygen consumption (Table 3). Although either NBT or vitamin E partially inhibited glucose oxidation, a combination of the agents was more potent than either one alone and was used in the subsequent studies. Unstimulated control PNIN treated with NBT-vitamin-E had rates of glucose oxidation similar to control cells which had not been exposed to the two agents. Following stimulation with HKB, control PMN treated with NBT-vitamin-E increased their rate of glucose oxidation only twofold compared with a 10-fold increase observed for untreated PMN1. The rate of glucose oxidation by CGD PMN in the presence or absence of NBT vitamin-E was decreased and not affected by the addition of HKB to the test svstem.

NBT-VITAMIN-E AND PMN FUNCTION

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Vol. 90, No. 3 March 1978

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Ultras_uctire

NBT and vitamin E alone or together at the highest concentrations employed in this study had no apparent effect on PMN (Figure 1). The surface membranes, cytoplasmic structure, storage organelles, and nuclei of treated PMN were well preserved and appeared identical to the morphologic features of untreated cells. PMN exposed to NBT-vitamin-E prior to stirring with bacteria readily ingested microorganisms into phagocytic vacuoles (Figure 2). The capacity of treated cells to phagocytose and incorporate bacteria appeared identical to the same processes in untreated PMN stirred with microorganisms for similar intervals. However, the phagocytic vacuoles containing bacteria in treated cells differed strikingly from those observed in PMN which were not exposed to NBT-vitamin-E. Bacteria in cells incubated with NBT and vitamin E were invested by tight-fitting vacuolar membranes, and the cytoplasm was filled with lysosomal organelles. Evidence of degranulation was virtually absent. In untreated cells the phagosomes were markedly distended, nearly replacing the cytoplasm, and storage granules appeared to be reduced in number (Figure 3). Portions of the same samples stained for myeloperoxidase activity also revealed a marked difference between NBT-vitamin-E treated PMN and untreated cells after ingestion of bacteria. The number of intact lysosomes reactive for the enzyme was greatly reduced in the untreated cells (Figure 4). Reaction product was evident in many phagocytic vacuoles but was absent from others, possibly due to leakage of enzyme to the surrounding medium. In contrast, PMN which had been incubated with NBT-vitaminE before stirring with bacteria retained large numbers of peroxidasepositive organelles in their Iytoplasm (Figure 5). Many phagosomes containing bacteria did not reveal peroxidase activity, while other vacuoles tightlv enclosing organisms contained small amounts of reaction product. Neutrophils from the patients with CGD closely resembled normal PMN which had been treated with NBT-vitamin-E. Untreated CGD neutrophils and those incubated with NBT-vitamin-E took up bacteria normally. Bacteria were enclosed by tight-fitting vacuoles in treated and untreated CGD cells, and large numbers of peroxidase-positive granules were retained in their cytoplasm (Figure 6). Peroxidase activity was evident in phagocytic vacuoles, indicating that degranulation was not completely blocked. Discssion Results of the present investigation have demonstrated that a combination of nitroblue tetrazolium and vitamin E strongly inhibits the oxidative

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response and degranulation associated with the uptake of particulates by normal polymorphonuclear leukocytes. The influence of NBT-vitamin-E on neturophil physiology did not appear related to cell damage, since incubation with the two agents at the highest concentrations used in this study produced no effect on ultrastructure. In fact, PMN exposed to NBT-vitamin-E appeared better preserved than cells which were untreated for similar intervals. In addition, NBT and vitamin E did not depress the phagocytic activity of PMN as assessed by light and electron microscopy. NBT has been used extensively in biologic studies to detect the activation of oxidative enzyme systems. When the yellow oxidized form of the dye is exposed to reducing equivalents, it is converted to a blue insoluble formazan.13 The agent is routinely used to detect the oxidative enzvme activity of PMN. When added to a suspension of neutrophils during phagocytosis, NBT is taken up in the vacuoles, where it is reduced to a blue precipitate.23 Dye reduction appears primarily due to superoxide anion generated from the oxygen consumed by the cell and reduced by oxidase-mediated electron flow from pyridine nucleotides.'3 In chronic granulomatous disease (CGD), a disorder in which patient PMN fail to develop the characteristic burst of oxygen consumption and activation of oxidative enzymes following uptake of bacteria, NBT taken up by the cells is not reduced.13'3 A considerable body of information regarding the mechanism whereby NBT is reduced during neutrophil phagocytosis has been developed. Our interest in examining its possible role as an inhibitor evolved from recent investigations of blood platelets and preliminary studies of lymphocytes. NBT was found to inhibit the response of platelets to aggregating agents and the effect of the dye was markedly potentiated by vitamin E.16 The combination also appears to block the response of lymphocytes to phytohemagglutinins.-' Results of these studies have suggested that NBTvitamin-E acts as a nonspecific scavenger system for free radicals generated during physiologic activation in a variety of cells.", The present study has demonstrated the potency of NBT and vitamin E in inhibiting neutrophil function. Appropriate concentrations of the two agents determined by repeated experiments caused marked depression of the respiratory burst in control PMN during ingestion of particulates. Levels of glucose oxidation and oxygen uptake by phagocytizing NBTvitamin-E-treated cells approached levels achieved by unstimulated control PMN or neutrophils from patients with CGD stimulated by bacteria. NBT alone and vitamin E alone partially inhibited the metabolic response

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of PMN, but the combination was more effective than either agent separately. Investigators have previously evaluated the effect of NBT or vitamin E on neutrophil function, but the combination of the two agents has apparently not been examined previously. Baehner and his associates found that NBT decreased by about half the oxygen consumption of activated PMN.'2 In another studv he and his colleagues observed that vitamin E increased phagocytosis and superoxide anion release by control PMN but decreased H202 production and bactericidal activity of the cells.u It is possible that the potentiation of vitamin E on inhibition of neutrophil function by NBT may be due to the transfer of electrons or protons to the dye, thereby facilitating reduction.' In addition to the marked influence on oxidative metabolism, the combination of NBT and vitamih E also had a striking effect on vacuole formation and the degranulation process in treated cells during uptake of bacteria. Distended vacuoles containing organisms nearly filled the cytoplasm of untreated cells, whereas the vacuolar membranes in NBTvitamin-E-treated cells fitted tightly around ingested organisms. Large numbers of myeloperoxidase-positive granules remained in treated cells, although some enzyme reaction product was evident in phagocytic vacuoles. In contrast, the cytoplasm of untreated cells was almost depleted of intact, peroxidase-positive organelles. Comparison of the response of NBT-vitamin-E-treated normal PMN during ingestion of bacteria to CGD neutrophils, whether exposed to the drug combination or not, revealed similarities. The NBT-vitamin-Etreated normal cells took up bacteria but failed to develop a burst of oxygen consumption, increased glucose oxidation, distended phagocytic vacuoles, and marked degranulation of lysosomes. All of these observations have been reported previously to be features of the defective response of CGD neutrophils.3'7 ' PMN from patients with CGD also fail to kill the ingested organisms, but this functional abnormality has not as yet been proved for the NBT-vitamin-E-treated normal neutrophil. Preliminary studies have demonstrated that NBT and vitamin E prevent bacterial growth in cultures. As a result, it has not been possible to assess the effect of NBT-vitamin-E on the capacity of neutrophils to kill bacteria bv ordinary methods. Other chemical compounds or combinations of agents have been found to depress PMN function and produce cells which mimic the defects of neutrophils from patients with CGD.' 31 The combination of NBT and vitamin E provides a new method of drug treatment for impairing normal

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neutrophil function. The defect observed in the NBT-vitamin-E-treated normal cells after ingestion of bacteria may resemble more closely the inherent abnormalities of CGD neutrophils than can be produced by any other agent or combination. Therefore, further studies to define the precise mechanisms through which NBT and vitamin E impair neutrophil function are warranted and are in progress. Refer 1. Hirsch JG: Cinemicrophotographic observations on granule lysis in polymorphonuclear leucocytes during phagocytosis. J Exp Med 116:827-834, 1962 2. Bainton DF: Sequential degranulation of the two tvpes of polymorphonuclear leukocyte granules during phagocytosis of microorganisms. J Cell Biol 58:249-264, 1973 3. Holmes B, Page AR, Good RA: Studies of the metabolic activity of leukocvtes from patients with a genetic abnormality of phagocvtic function. J Clin Invest 46:14221432, 1967 4. Babior BM, Curnutte JT, McMurrich BJ: The particulate superoxide forming system from human neutrophils. Properties of the system and further evidence supporting its participation in the respiratory burst. J Clin Invest 58:996, 1976 5. Babior BM, Kipnes RS, Curnutte JT: Biological defense mechanisms: The production by leukocytes of superoxide, a potential bactericidal agent. J Clin Invest 52:741744, 1973 6. Tauber Al, Babior BM: Evidence for hvdroxyl radical production by human neutrophils. J Clin Invest 60:374-379, 1977 i. Klebanoff SJ: Intraleukocvtic microbicidal defects. Annu Rev Med 22:39-62, 1971 8. Quie PG, White JG, Holmes B, Good RA: In vitro bactericidal capacitv of human

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11. 12.

13. 14.

15. 16. 17.

polymorphonuclear leukocytes: Diminished activity in chronic granulomatous disease of childhood. J Clin Invest 46:668-679, 1967 Klebanoff SJ: Myeloperoxidase contribution to the microbicidal activitv of intact leukocytes. Science 169:1095-1097, 1970 Spitznagel JK, Cooper MR, McCall CE, DeChatelet D: Selective deficiency of granules associated with Iysozyme and lactoferrin in human polvmorphonuclear leukocytes (PMN) with reduced microbicidal capacity. J Clin Invest 59:93a, 1972 Curnutte JT, Whitten DM, Babior BM: Defective superoxide production by granulocytes from patients with chronic granulomatous disease. N Engl J Med 290:593597, 1974 Baehner RL, Murrmann SK, Davis J, Johnston RB Jr: The role of superoxide anion and hydrogen peroxide in phagocytosis-associated oxidative metabolic reactions. j Clin Invest 56:571-576, 1975 Baehner RL Boxer LA, Davis J: The biochemical basis of nitroblue tetrazolium reduction in normal human and chronic granulomatous disease polymorphonuclear leukocytes. Blood 48:309-313, 1976 Curnutte JT, Kipnes RS, Babior BM: Defect in pyridine nucleotide dependent superoxide production by a particulate fraction from the granulocvtes of patients with chronic granulomatous disease. N Engl J Med 293:628-632, 1975 Briggs RT, Karnovsky ML, Karnovsky MJ: Hydrogen peroxide production in chronic granulomatous disease. J Clin Invest 59:1088-1098, 1977 White JG, Rao GHR, Gerrard JM: Effects of nitroblue tetrazolium and vitamin E on platelet ultrastructure, aggregation, and secretion. Am J Pathol 88:387-402, 1977 Repine JE, Clawson CC: Quantitative measurement of the bactericidal capabilitv

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20. 21. 22.

23. 24. 25. 26.

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of neutrophils from patients and carriers of chronic granulomatous disease. J Lab Clin Med 90:522-528, 1977 Repine JE, White JG, Clawson CC, Holmes BM: Effects of phorbol myristate acetate on the metabolism and ultrastructure of neutrophils in chronic granulomatous disease. J Clin Invest 54:83-90, 1974 Repine JE, Clawson CC, Friend PS: Influence of a deficiency of the second component of complement on the bactericidal activity of neutrophils in vitro. J Clin Invest 59:802-89, 1977 White JG: Fine structural alterations induced in platelets by adenosine diphosphate. Blood 31:604-622, 1968 White JG: Interaction of membrane systems in blood platelets. Am J Pathol 66:295-312, 1972 Graham RC Jr, Kamovsky MJ: The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: Ultrastructural cytochemistry by a new technique. J Histochem Cytochem 14:291-302, 1966 Nathan DG, Baehner RL, Weaver DK: Failure of nitroblue tetrazolium reduction in the phagocytic vacuoles of leukocytes in chronic granulomatous disease. J Clin Invest 48:1895-1904, 1969 Rao G, Beall GD, White JG: Unpublished observation Baehner RL, Boxer LA, Allen JM, Davis J: Autooxidation as a basis for altered function by polymorphonuclear leukocytes. Blood 50:327-335, 1977 Michaelis L, Wollman SH: The semiquinone radical of tocopherol. Science

109:313-314, 1949 27. Baehner RL, Karnovsky MJ, Karnovsky ML: Degranulation of leukocvtes in chronic granulomatous disease. J Clin Invest 47:187-192, 1968 28. Gold SB, Hanes DM, Stites DP, Fudenberg HH: Abnormal kinetics of degranulation in chronic granulomatous disease. N Engl J Med 291:332-337, 1974 29. Holmes B, Good RA: Laboratory models of chronic granulomatous disease. J Reticuloendothel Soc 12:216-237, 1972 30. Malawista SE, Bodel PT: The dissociation by cholchicine of phagocytosis from increased oxygen consumption in human leukocytes. J Clin Invest 46:786-796, 1967 31. Mandell GL, Rubin W, Hook EW: The effect of an NADH oxidase inhibitor (hydrocortisone) on polvmorphonuclear leukocyte bactericidal activitv. J Clin Invest 49:1381-1388, 1970

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Figure 1-Normal polymorphonuclear leukocyte (PMN) from a sample of neutrophils fixed in glutaraldehyde and osmic acid after incubation with the highest concentrations of nitroblue tetrazolium (NBT) and vitamin E used in this study for 1 hour. Morphologic features of the PMN are well preserved after exposure to the two agents. Nuclear (N) chromatin and parachromatin are distributed normally. Azurophilic lysosomes (L) and specific granules (G) fill the cytoplasm. No evidence of swelling, loss of organelles, rounding up, or cytoplasmic injury is apparent. A Golgi zone (GZ) consisting of parallel, flattened saccules is also an indication that the cell has not been damaged. Glycogen particles are distributed randomly in the cytoplasm. Neutrophils exposed to NBT or vitamin E alone for a similar period were also well preserved. (Uranyl acetate and lead citrate, X14,900)

Figure 2-The normal neutrophil in this illustration is from a sample of PMN incubated with NBT and vitamin E and then stirred with bacteria. Organisms (arrows) engulfed by the cell are enclosed within tight-fitting membranes. No evidence of phagocytic vacuole distention or degranulation of cytoplasmic organelles is evident in the PMN. (Uranyl acetate and lead citrate, x 16,500)

C.

i,.

I

1. .

-4.

Fgure 3-Normal PMN stirred with bacteria for a similar period of time under conditions idenbcal to those employed for the sample shown in Figure 2. However, the neutrophils had not been incubated with NBT-vitamin-E prior to addition of the organisms. Bacteria taken up by the cell are in distended phagocytic vacuoles (V) and residual cytoplasm is virtually devoid of granules. (Uranyl acetate and lead citrate, x 16,500)

FiLgwe 4-Normal PMN from a sample of neutrophils fixed after stirring with bacteria and incubated with diaminobenzidine and hydrogen peroxide to demonstrate myeloperoxidase. The cells were not exposed to NBT-vitamin-E prior to addition of the organisms. Reaction product is evident around the bacteria in phagocyfic vacuoles (V), but only a few enzymepositve lysosomes (L) remain in the cytoplasm. (x 19,000)

3

I

Fgure 5-Neutrophil from a sample of PMN treated with NBT-vitamin-E before exposure to bacteria and stained for myeloperoxidase after fixation. Reaction product is evident around some bacteria (arros), but the vacuoles are not distended. A large number of intact lsosomes posftive for enzyme reaction product remain in the cytoplasm of the cell. (x 15,000) Fre 6-Neutrophil from a patient with chronic granulomatous disease. The abnormal PMN sample

was stirred with bacteria without prior incubation with NBT and vitamin E, conditions identical to those used for the neutrophil sample shown in Figure 4. Reaction product is evident inside the tight-fitting phagocytic vacuoles (arrows), but large numbers of peroxidase-positive granules remain in the cytoplasm. (Uranyl acetate or lead citrate, x 17,000)

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