Effect of blitro Blue Tetrazolium Dye on the Hexose Monophosphate Shunt Activity of Human Polymorphonuclear Leukocytes LAWRENCE
PAMELA S. SHIRLEY
The Department of Biochemistry. The Bow,man Gray School of Medicine, Winston-Salem. North Carolina 27103 Received
During the process of phagocytosis, human polymorphonuclear leukocytes (PMNL) exhibit marked alterations in oxidative metabolism, including increases in oxygen consumption, hexose monophosphate shunt activity, and hydrogen peroxide production and the production of very reactive species of molecular oxygen including superoxide anion and singlet oxygen (1). If normal PMNL are allowed to phagocytize in the presence of the redox dye, nitro blue tetrazolium (NBT), the dye is observed to be reduced to the insoluble blue formazan within the cell (2). Although the precise basis for these alterations in metabolism is uncertain, there is general agreement that activation of an oxidative enzyme would provide a reasonable explanation. Baehner and Karnovsky (3) have favored NADH oxidase as the critical enzyme, based primarily on their observation that the activity of this particular enzyme is reduced in the defective cells obtained from patients with chronic granulomatous disease of childhood. Others, however, have not been able to verify this observation (4), and some workers have proposed that NADPH oxidase is the initiating enzyme (5,6). Two recent reports (7,8) have suggested that this enzyme, and not NADH oxidase, is defective in cells obtained from patients with chronic granulomatous disease. The situation is presently far from settled. In a recent paper, Wilkinson rt al. (9) studied the effects of NBT dye on the post-phagocytic metabolism of human PMNL. Under their experimental conditions, the addition of 0.1% NBT abolished the increases in oxygen consumption and hexose monophosphate shunt activity usually associated with phagocytosis. The authors used these data to argue that NADH oxidase rather than NADPH oxidase was the initiating enzyme of the respiratory burst. We have been investigating the same phenomenon, but our results are quite different from those reported by Wilkinson et al. (9). In the present paper, we present data which strongly suggest that the inhibition of hexose monophosphate shunt activity by NBT is due to an inhibition of 391 Copyright @ 1975 by Academic Press. Inc. All rights of reproduction in any form reserved.
phagocytosis and/or a decrease in cell viability under the assay conditions employed. Indeed, it is possible to select other conditions under which the addition of NBT results in a stimulation of hexose monophosphate shunt activity in phagocytizing human PMNL. Thus, the experiments of Wilkinson clt al. (9) cannot be interpreted as favoring either NADH or NADPH oxidase as the initiating enzyme of the respiratory burst. METHODS Human PMNL were isolated from 50 ml of heparinized venous blood by sedimentation of the erythrocytes with plasma gel as previously described ( 10). The final cell suspension, consisting of 80-90% PMNL, was suspended in Dulbecco’s phosphate-buffered saline to a final concentration of 5x 10” PMNLiml. Preparation of NBT solutions. The predetermined quantity of nitro blue tetrazolium (Sigma Chemical Co., St. Louis, Missouri) was dissolved in 0.15 M sodium phosphate buffer, pH 7.4. This was diluted with an equal volume of 0.9% NaCl to yield a stock solution of 1% NBT. The stock solution was prepared fresh on the day of use and was filtered immediately before use to remove the small amount of insoluble material which was invariably present. A control solution, consisting of equal amounts of phosphate buffer and 0.9% NaCl. was routinely added to all control incubation flasks instead of the NBT. Mcasurrr?lrnt of hr.uosr nlonoph~)sphrrtc~ shunt ac,titity. Glucose utilization via the hexose monophosphate shunt was determined by a modification of the procedure previously described ( I 1). Each flask contained 1.O ml of cell suspension (5x 10” PMNL/ml of PBS); 0.30 ml of pooled human serum (in some experiments this was replaced by 0.30 ml of 100 mg/dl of glucose solution); 0.05 $Zi of [I-14C]glucose (New England Nuclear Corp., Boston, Massachusetts); and NBT solution or phosphate-saline buffer as indicated. Phagocytosis was initiated in appropriate flasks by the addition of 0.20 ml of standard latex suspension (I 1). Volumes of all flasks were adjusted to 3.0 ml by the addition of an appropriate quantity of PBS. Reaction was initiated by the addition of the cell suspension, was allowed to proceed for the stipulated period of time at 37”. and was terminated by the addition of 1 .O ml of 10% trichloroacetic acid. ‘“CO, liberated during the course of the incubation was collected in a center well containing 0.5 ml of hyamine hydroxide and counted in a liquid scintillation spectrometer as previously described (I I ). Mrasurc~rnrnt o$phagocytosis. Radiolabeled bacteria were prepared by inoculating 10 ml of trypticase soy broth containing 100 &i of a mixture of uniformly ‘“C-labeled amino acids (New England Nuclear Corp.) with Eschrric~hirr c.oli B. After incubation at 37” for 24 hr, the bacteria were killed by heating in a boiling-water bath for 20 min. The killed bacteria were Zsol~~tiorz c!f’ Idmytc~s.
NBT AND LEUKOCYTES
collected by centrifugation at 27,000 g, washed three times with 0.9% NaCl, and suspended in 3.0 ml of 0.9% NaCl. The bacterial suspension was opsonized by the addition of 6.0 ml of normal human serum followed by incubation for 30 min at 37”. After opsonization, the bacteria were collected by centrifugation and resuspended in PBS to a standard absorbance of 0.08 at 525 nm on a Coleman Junior spectrophotometer. For the phagocytic assay: a 1.O-ml amount of leukocyte suspension was incubated with 0.30 ml of glucose solution (100 mg/dl), 0.30 ml of 1% NBT solution (or the buffer in which the NBT was dissolved), and 1.O ml of the standard bacterial suspension: volumes were adjusted to 3.0 ml by the addition of the appropriate amount of PBS. The reaction was stopped after varying periods of time by the addition of 4.0 ml of cold 0.04 M sodium fluoride in PBS. The cells were collected by centrifugation at 800 g for 10 min, and the pellets were washed free of non-ingested bacteria by repeated suspension in 5.0 ml of 0.01 M sodium fluoride in PBS containing 10% fetal calf serum. After three such washes, the pellet was dried overnight in a 60” water bath and digested in 0.50 ml of 0.2 N NaOH for 4 hr. The solutions were then neutralized with 0.20 ml of 3% glacial acetic acid, 0.50 ml of deionized water was added, and 1.O-ml volumes were counted in 10 ml of Aquasol (New England Nuclear Corp.) with a liquid scintillation spectrometer. Assaysfor Cell viability. Cell viability was assayed in two ways. In the first procedure, the cells were incubated in the presence of either 0.1% NBT or the corresponding buffer. Samples were removed at varying periods of time and diluted 1:20 with a fresh solution of 0.2% trypan blue dye in 0.9% NaCl, using a white blood cell diluting pipet. The cells were examined in a hemacytometer and the percentage of cells which excluded the dye was taken as an indication of cell viability. This procedure works well with control cells, but, in the presence of 0.1% NBT dye, extensive clumping of the cells occurs with substantial adherence to the walls of the vessel. Thus the sample examined by this method in the presence of NBT cannot be considered representative. Further, only cells which remain intact during the incubation can be counted by this procedure; cells which have lysed or disintegrated cannot be measured. Because of the above-mentioned limitations, we also assessed viability by measuring the release of a cytoplasmic marker enzyme, lactic dehydrogenase (LDH), into the incubation medium. Cells were incubated with either 0.1% NBT dye or control buffer for varying periods of time. The cells were separated from the supernatant by centrifugation at 27,000g for 15 min and the supernatants assayed for LDH by a standard spectrophotometric method (12). Values for total cellular LDH were obtained by sonically rupturing a similar quantity of cells. Phase microscopy indicated that the sonication resulted in complete cell breakage; similar values
for total LDH were obtained regardless of whether the cells were disrupted in the presence or absence of 0.1% NBT. This procedure likewise has limitations in that only cells which have been damaged to the point of leakage of enzymes will contribute to the supernatant LDH: thus it too may represent a low estimate of cellular death. RESULTS Table 1 illustrates the lack of effect of 0.40 mg/ml(O.O4%) of NBT on the hexose monophosphate shunt activity of human PMNL. These experiments utilized an incubation time of 60 min and were run in the presence of 10% pooled normal serum. The addition of NBT appears to cause a slight stimulation of glucose oxidation in resting cells. The addition of latex particles to resting cells results in a marked stimulation of hexose monophosphate shunt activity which is completely unaffected by the presence of NBT dye. If the post-phagocytic hexose monophosphate shunt (HMS) activity is measured under the same conditions at very short time intervals, a stimulation of activity by NBT is observed (Fig. 1). This stimulation is greatest at the shortest time intervals and becomes progressively less apparent as the time of incubation is prolonged; it is not observed at time intervals as long as 60 min (Table 1). These data are quite different from those reported by Wilkinsonc’t al. (9). In an attempt to reconcile the disparate results. we repeated the assay using 0.1% NBT in the absence of serum (substituting an equivalent amount of 100 mg/dl of glucose). Under these conditions, we observed a profound inhibition of the phagocytizing shunt activity during the course of a 60-min incubation (data not shown), in complete agreement with the data preTABLE EFFECT
I F SHUN
I .4c FIVI I 1 OI
E.~R L~r:~ocy~rs” .~ Hexoae
Resting cells (5) + NBT (5) Phagocytizing cells
+ NBT ‘* Values individual was added,
represent the means f SEM. Numbers experiments. Each value was determined where indicated. to a final concentration
1.984 17.843 17.682
? 338 c 1.092 k 1.114
in parentheses represent the number of in triplicate in each experiment. NBT of 0.40 mg/ml.
NBT AND LEUKOCYTES
Time ( mm 1
FIG. 1. Hexose monophosphate shunt activity of human PMNL following phagocytosis of latex particles in the presence of serum. Open bars, control; hatched bars, 0.40 mg/ml of NBT present. The data are taken from a single experiment which is representative of four separate experiments; each point was determined in triplicate in each experiment.
viously published. In the absence of serum even very low concentrations of NBT result in some inhibition of HMS activity (Fig. 2). The highest concentration of NBT used in this experiment was fourfold lower than that employed by Wilkinson ef al. (9). Other experiments (not shown) demonstrated that 1 mg/ml of NBT inhibited the phagocytizing HMS activity at short time intervals in the absence of serum. Lower concentrations of NBT gave progressively less inhibition, but in no case was an actual stimulation of glucose oxidation observed when serum was omitted. In an attempt to determine whether the dye might be inhibiting
FIG. 2. Effect of NBT concentration on phagocytizing HMS activity of human polymorphonuclear leukocytes in the absence of serum. Each point is the mean of closely agreeing triplicate determinations.
phagocytosis in the absence of serum, we examined the effects of I mg/ml of NBT on the uptake of radiolabeled bacteria by PMNL. Results are seen in Fig. 3. Even at the shortest time interval examined, there is a marked suppression of bacterial phagocytosis by the dye. This inhibition is virtually complete by 5 min, and very few additional bacteria are ingested between 5 and 15 min. in contrast to the results seen in the absence of NBT. We attempted to assess the influence of 0.1% NBT on cell viability by monitoring the ability of the cells to exclude 0.2% trypan blue dye. During the course of incubation there was a progressive loss of viability in the presence of 0.1% NBT so that, by 60 min, only 64% of the cells examined were viable by the trypan blue exclusion test; in contrast 99% of the cells in the control incubation were viable by this criterion after 60 min. There were far fewer cells present in the samples removed from the incubation containing the NBT, and extensive clumping was observed in the bottom of the flask. Thus it was considered likely that the actual viability was considerably less than that observed; the cells which suffered the greatest damage in the presence of the dye (by extensive clumping or even Iysis) were excluded from the sample taken. Figure 4 illustrates the release of the cytoplasmic marker enzyme. LDH, into the supernatant fraction as a function of incubation time. A relatively small amount of enzyme t- 5%) is released from the control cells: this release is relatively constant over the time period examined. In contrast, cells incubated in the presence of 1 mg/ml of NBT release significantly more LDH at all time periods examined: the amount released increases with incubation time, so that by 60 min nearly half of the total cellular enzyme may be found in the supernatant.
FIG. 3. Effect of NBT on phagocytosis of radiolabeled /-..