BritishJournal ofHuernatology, 1975,31, 193.
Stimulation by Propylthiouracil of the Hexose Monophosphate Shunt in Human Polymorphonuclear Leucocytes during Phagocytosis MIN-FU TSAN AND PATRICIA A. MCINTYRE Division of Hematology, Department of Medicine, The Johns Hopkins Medical Institutions, Baltimore, Maryland (Received 7 February 1975 ; accepted for publication 13 March 1975) SUMMARY. The effect of propylthiouracil on glucose metabolism in human polymorphonuclear leucocytes was studied. At a therapeutically achievable concentrapropylthiouracil , stimulated hexose monophosphate shunt activity tion (0.I a) in normal leucocytes during phagocytosis but not in resting cells. However, in the presence of hydrogen peroxide it stimulated hexose monophosphate shunt activity in resting cells, and in the soluble fraction when reduced glutathione and reduced nictotinamide adenine dinucleotidephosphate (NADPH) were also present. Propylthiouracil had no effect on glucose-I-C oxidation in either phagocytosing or resting leucocytes obtained from two male patients with chronic granulomatous disease. Stimulation of the hexose monophosphate shunt activity in normal leucocytes during phagocytosis also was demonstrated with methimazole, thiouracil and thiourea, but not with adenine, uracil or urea. There was an apparent minimal common structure requirement in thiourea. Propylthiouracil had no effect on phagocytosis, formate oxidation, glucose-&phosphate dehydrogenase, 6phosphogluconate dehydrogenase or catalase activities. Thus, the stimulation of the hexose monophosphate shunt activity by propylthiouracil is dependent on hydrogen peroxide and is best explained by its stimulation or participation in the glutathione cycle. Agranulocytosis is the most serious side effect encountered during treatment of hyperthyroidism with thiouracil and related compounds (Bartels, 1948 ; Trotter, 1962). Despite this devastating but fortunately rare complication, these drugs continue to enjoy wide clinical use. The rare occurrence and the lack of correlation between this complication and the dosage or duration of therapy, suggest that something other than a direct toxic effect of these drugs is responsible for the development of agranulocytosis. In fact, recently, evidence has been presented indicating that an immune mechanism may be responsible for this potentially fatal complication (McIntyre et al, 1971 ; Bilezikian et al, 1973). Our knowledge about the direct effect of these antithyroid drugs on normal leucocytes is fragmentary. Thiouracil was found to inhibit oxygen consumption in human (McCurrach et al, 1970) and rabbit (Warren, 1945) polymorphonuclear leucocytes (PMN). Martelo et a1 (1967) fiiled to demonstrate any effect of propylthiouracil (PTU) on DNA, RNA or protein Correspondence: Dr Patricia A. McIntyre, 615 N. Wolfe Street, Baltimore, Maryland 21205,U.S.A.
Min-Fu Tsan and Patricia A. McIntyre I94 synthesis in human bone marrow cells. Reed & Tepperman (1969) showed that peritoneal exudate PMN from rats, in which hypothyroidism had been induced by PTU, exhibited increased rates of oxygen consumption and oxidation of glucose-I-C and glucose-6-C in both resting cells and cells during phagocytosis. During the course of previous investigations of antithyroid drug induced agranulocytosis, it was found that PTU at a relatively high concentration (1.7 m ~stimulated ) C 0 2production from glucose-I-C in normal human PMN during phagocytosis (McIntyre et al, 1971). In this study, we have demonstrated that PTU, at a therapeutic concentration, caused stimulation of the hexose monophosphate shunt (HMS) activity in human leucocytes during phagocytosis but not in resting cells. Leucocytes obtained from patients with chronic granulomatous disease failed to show this effect. Evidence is provided that the effect was not mediated by increasing the phagocytic capacity of the PTU treated cells but was dependent on hydrogen peroxide (H202).Participation of PTU in the glutathione cycle is the most likely explanation of this effect.
MATERIALS AND METHODS Chemicals. [~-‘~C]glucose,[6-14C]glucose and [I4C]formate were obtained from Amersham/Searle Corp., Arlington Heights, Ill. Thiouracil and 6-propyl-2-thiouracil (PTU) were obtained from Nutritional Biochemicals Corp., Cleveland, Ohio. Thiourea, urea, adenine, uracil, I-rnethyl-imidazole-thiol (methimazole), HzOz(as a 30% solution), reduced glutathione (GSH) and nicotinamide adenine dinucleotide phosphate (NADP) were obtained from Sigma Chemical Co., Indianapolis, Ind. Crystalline beef liver catalase was obtainedfrom Boehringer and Mannheim, Germany. Isolation of human polymorphonuclear leucocytes. Human polymorphonuclear leucocytes were obtained from normal donors and from two male patients with chronic granulomatous disease who were followed at the National Institutes of Health, Bethesda, Md. Briefly, 50-100 ml of venous blood was drawn from normal individuals or patients into 17.5-35 ml of a solution that contained 120mM glucose, 28 m~ EDTA, 4.2% Dextran-70 (Cutter Labs., Berkeley, CaliE) and 170 m~ NaCI. After gentle mixing, the red cells were allowed to settle in tubes placed in ice for 60 min. The leucocyte-rich plasma was collected into 50 ml plastic tubes and centrifuged at 200 g at 4OC for 10 min. The supernatant was discarded and the pellet was suspended in 0.83% NH,C1 to lyse the contaminating red cells. The cell suspension was then centrifuged at 80 g for 10 min at 4°C. The final cell pellet was suspended in modified Hanks’ solution containing 5 m~ glucose as described previously (Tsan & Berlin, 1g71a). With this method approximately 160-57ox 106 leucocytes can be obtained from IOO ml of venous blood. The preparation contained about 90% granulocytes and was relatively free of platelets as determined by examination with phase contrast microscopy. The remaining cells were primarily lymphocytes and monocytes. Metabolic studies. For the collection of [‘4C]carbon dioxide, the metabolic flask (Kontes Glass Co., Vineland, N.J.) described by Saba & Di Luzio (1966)was used. A liquid scintillation counting vial containing a g x 3 cm strip of filter paper soaked with 0.5 ml of 10% NaOH was connected to the side arm of this metabolic flask with a Teflon adapter. To each flask a final volume of 2 ml of modified Hanks’ solution (5 m~ glucose), containing 10x 106 cells,
Propylthiouracil and Polymorph Function
I95 2 m~ Mg' +, and radiolabelled glucose or formate in the presence or absence of particles, sera or chemicals to be tested, was added. The radioactivity used in each flask was z x 105 dpm in case of [~-'~C]glucose and 3.3 x 105dpm in case of [614C ]gl~~ose and ['4C]formate. The final concentration of particles was 0.17% by weight and that of serum was 10%when used. Preliminary experiments revealed that similar results were obtained with either 0.79 pm polystyrene latex particles, or 2.02 pm polyvinyl toluene latex particles. Subsequently, all metabolic studies were done with 0.79 pm polystyrene latex particles. After addition of the incubation medium, the flask was covered immediately with an air tight rubber stopper and incubated in a shaking water bath at 37°C for 60 min. The reaction then was terminated by injecting I ml of 62.5% citric acid through the rubber stopper. Incubation was continued for another 60 min to ensure maximum absorption of [14C]carbon dioxide by the filter paper. The vials were removed and dried overnight in a vacuum dessicator and prepared for counting by liquid scintillation (1000 ml toluene and 42 ml Liquifluor, New England Nuclear Corp., Boston, Mass.). The dpm were calculated by correcting the quenchmg effect based on channel ratio (Herberg, 1965). In each experiment samples were done in duplicate and the results were averaged. When leucocytes were not present in the incubation medium, the radioactivity recovered from glucose was negligible. However, in case of ['4C]formate there was a fixed amount of radioactivity recovered upon addition of acid, consequently a correction was made in each experiment with ['4C]formate. Analysis of variance (Federer, 1955; Snedecor& Cochran, 1967) was used to assess the statistical significance. Quantijication of phagocytosis. Quantitative measurement of phagocytosis was performed as described by Tsan & Berlin (1g71b), based on the method of Roberts & Quastel (1963). Briefly, cell monolayers (2x 106PMN per coverslip) were made on 22 mm diameter coverslips by incubating 0.5 ml aliquots of cell suspensions for 30 min at 37°C. After the formation of monolayers, the coverslips were drained and aliquots of approximately 0.4 ml of incubation media containing 0.2% by weight of polyvinyl toluene latex particles (2.02 pm in diameter), 2 m~ Mg' +,10% serum, and chemicals to be tested in modified Hanks' solution (5 mM glucose), prewarmed to 37°C were placed over the monolayers. After incubation for 30 min, the coverslips were drained and rinsed .consecutively in four beakers containing ice cold isotonic sucrose solution. The coverslips were placed in 20 ml beakers containing 2 ml dioxane, and extracted overnight. The extracted polyvinyl toluene was measured with a Beckman spectrophotometer (National Technical Lab., South Pasadena, Calif.) at 274 nm. The controls were dioxane extracts of monolayers incubated with the same media at 0°C for 30 min. Phase contrast microscopy revealed that after 30 min incubation at 37°C in the absence of serum, there were occasional clumps of particles which could not be eliminated by the rinsing procedure. This phenomenon was particularly prominent with 0.79 pm polystyrene latex particles. In the presence of 10% serum, this contamination with latex particles were completely eliminated by using 2.02 pm polyvinyl toluene latex particles. When incubated at 0°C only some particles were found adherent to the glass and to the cell periphery. Few could be identified intracellularly by phase contrast microscopy and consequently the 0°C value was subtracted from that obtained at 37°C in the presence of 10% serum to calculate the amount of particles actually taken up by the cells. Preparation ofcellfractions. After collection of leucocytes, the pellet was washed twice with modified Hanks' solution and resuspended in alkaline isotonic potassium chloride (0.32 ml
Min-Fu Tsan and Patricia A. McIntyre
of 0.1 M KHCO, in
nil of 154 mM KCl) as a 20% cell suspension (Noseworthy & Karnovsky, 1972). For the measurement of direct oxidation of [~-'~C]glucose,the cells were homogenized in ice for 5 min in a glass homogenization tube fitted with a Teflon pestle on an electrically driven homogenizer (Noseworthy & Karnovsky, 1972). Cell counting revealed that about 60-70% of cells were disrupted by this procedure. The homogenate then was centrifuged at 510 g at 4°C for 10 min to remove cell debris and unruptured cells. The supernatant contained both granule and soluble fractions and was designated as 'homogenate fraction'. Part of this homogenate fraction was centrifuged at 20 o w g for 20 min at 4°C to obtain the 'soluble fraction'. Both homogenate fraction and soluble fraction were used for the measurement of HMS activity. For the measurement of glucose-&phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities, leucocytes were preincubated with and without particles in the presence and absence of PTU for 15 min at 37°C. The cells were spun down at 60 g at 4°C for 10 min. The pellets were washed twice with modified Hanks' solution and resuspended in alkaline isotonic potassium chloride. The cells were disrupted by freezing and thawing three times, using a mixture of dry ice and methanol (Ramot et al, 1959). Leucocyte counts were performed before and after this procedure. No more than 5% of cells remained intact. The leucocyte extracts were again centrifuged at 20 ooo g at 4°C for 20 min and the supernatant was used for enzyme determinations. Enzyme assays. The activities of glucose-&phosphate dehydrogenase and 6phosphogluconate dehydrogenase of the soluble fraction were assayed according to the method of Glock & McLean (1g53), modified as follows: I mlo.19 M tris buffer, pH 8.0; 0.1 mlo.3 M Mg' ; 0.1 ml 20 niM glucose-&phosphate; 0.1 ml 20 mM 6-phosphogluconate; 0.2 ml z niM NADP' ;0. I ml of soluble fraction (from 2 x 106WBC) and distilled water to produce a total volume of 3 ml in a I cm light path cuvette. The change in optical density at 340 nm at room temperature was followed with time in the Beckman Spectrophotometer, equipped with an automated multiple sampling and continuous recording system (Gilford Model 2000, Multiple Sample Absorbance Recorder, Oberlin, Ohio). The reaction was linear for at least 5 min. The activity of G6PD was derived from the difference between the activities measured with 6phosphogluconate alone (6PGD activity) abd the combination of glucose-6-phosphate and 6phosphogluconate. For the purpose of comparison with the data of Beck (1958)~ the results were expressed inpmoles NADP+ reduced per min per IO'O cells. The effects of PTU and KCN on crystalline beef liver catalase were assayed according to Bergmeyer (1955),modified as follows: 0.1 ml 3% H 2 0 2; 10 p1 catalase (containing 0.2 pg catalase) and 0.05 M phosphate buffer pH 6.8 to make a final volume of 3 ml in a I cm light path cuvette. Experimental additions to this basic system were as follows: 0.1 ml 5 niM or 0.5 mM PTU, 0.1 ml 50 mM or 5 mM KCN. The change in optical density at 240 nm at room temperature was followed spectrophotometrically as described above. The reaction was linear for at least 10s. Propylthiouracil at the concentrations used had some absorption at 240 nm, causing a slight elevation of the baseline recording, but it did not interfere with the measurement of the changing concentration of H 2 0 2after addition of catalase. Measurement of HMS in cell-free fractions. In general the incubation was carried out as described in Metabolic Studies. The incubation system employed was a modification of that used by Beck & Valentine (1952). Final concentrations of components were as follows: 2 mM
Propylthiouracil and Polymorph Function
Mg' ; 2 mM glucose (0.4PCi [~-'~C]glucose); 2.4 mM ATP and I ml of homogenate or soluble fraction (containing 0.5-0.6 mg protein in soluble fraction). Experimental additions to this basic system yielded the following final concentrations: 0.3 n m NADPH; 0.8 mM PTU; 4 mM GSH; 5.2 mM HzOz and 0.8 niM KCN. Total volume was 2.5 ml in each flask. Protein determination. Determinations of the protein content of soluble fractions were performed by the method of Lowry et a1 (1951). +
Eject of propylthiouracil (PTU) on glucose utilization in human P M N leucocytes. Glucose utilization was measured with [~-'~C]glucoseand [6-14C]glucose. As shown in Table I, in the absence of serum and PTU there was a three-fold stimulation, and in the presence of 10% serum there was a more than Io-fold stimulation of glucose-I-C oxidation .in cells during phagocytosis. Addition of propylthiouracil at a concentration of 0.I m~ produced an additional ~O-IOO% stimulation in the presence and absence of serum in cells during 100 -
Concentration (mw )
FIG I. Effect of propylthiouracil on glucose-l-C oxidation in human poiymorphonuclear leucocytes during phagocytosis. Each point represents the mean of two duplicate experiments.
phagocytosis but had no effect in resting cells. In contrast, PTU had no effect on glucose-6-C oxidation in either resting or phagocytosing PMN. Fig I shows the dose-response curve of the effect of PTU on glucose-I-C oxidation in cells during phagocytosis in the absence of serum. Even at 0.1 mM, PTU significantly stimulated CO, release from glucose-I-C. A marked stimulation of HMS as measured by CO, production from glucose-I-C during phagocytosis has been documented (Sbarra & Karnovsky, 195g), as also is evident from our results. However, such a further stimulation of HMS in the presence of a therapeutic concentration of propylthiouracil(0. I mM) during phagocytosis has not previously been reported. Therefore, investigations were carried out with other antithyroid drugs and structurally related substances to see whether this effect was shared by other compounds. Eiect ofother antithyroid drugs and related compounds on HMS in human PMN. The following
Min-Fu Tsan and Patricia A. McIntyre TABLE I. Effect of PTU on [14C]C02release from [14C]glucose by human PMN leucocytes*
Resting cells Control +PTU P value Phagocytosing cells Control +PTU P value Senun +Senun+PTU P value
* The results are expressed as meankstandard error of the mean (dpm). Each experiment was done in duplicate and the results were averaged. The radioactivity added to each flask was L O X 1oS dpm for [~-~~C]glucose and 3.3 x 105 dpm for [6J4C]glucose. The concentration used: PTU: 0.1 mM, serum: 10%. t Number in parentheses indicates number of experiments. OH
Loctim form of uracil
Lactam form of uracil
FIG 2. Chemical structures of propylthiouracil and its related compounds. The structure (thioureylene group) within the dashed line is the minimal common structure required for the stimulation of HMS in PMN leucocytes during phagocytosis.
Propylthiouracil and Polymorph Function
compounds were studied at the concentration of 0.I mM : methimazole, thiouracil, uracil, adenine, thiourea and urea. None of these compounds have any effect on COz production from glucose-carbon-1 in resting cells. However, during phagocytosis, methimazole, thiouracil and thiourea, as in the case of propylthiouracil, stimulated the HMS. Uracil, adenine and urea had no effect (Table 11). Some of the structure-function relationships are immediately apparent from these results (Fig 2). The propyl group at carbon-6 position of PTU is not essential since thiouracil shared the same effect. A substitution of the thiol group with ox);gen at the carbon-2 position abolished this effect as can be seen from uracil and urea (as compared to thiouracil and thiourea). The cyclic ring is also not required as can be seen from thiourea (as compared to thiouracil, methimazole and PTU). The minimal common structure from this study is thiourea (or a thioureylene group -NH-C-HN-).
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of PTU on leucocytesfrom patients with chronic granulomatous disease. Leucocytes from patients with chronic granulomatous disease have normal phagocytic capacity, but fail to show the usual stimulation of oxygen consumption, H z O z production and HMS activity during phagocytosis, with impaired intracellular bacterial killing of catalase positive bacteria (Holmes et al, 1967; Karnovosky, 1973). The PTU effect was tested on PMN from two male patients with chronic granulomatous disease and the results were shown in Table 111. It is apparent that leucocytes from these patients showed very low HMS activity and PTU had no effect in either resting cells or cells during phagocytosis. Efict ofPTU on phagocytosis of latex particles. Phagocytosis was studied with phase contrast microscopy and quantitative measurement by spectrophotometry as described above. Recent evidence (Rossi et al, 1972) suggests that the HMS can be stimulated by the interaction between particles and surface membrane of leucocytes.Therefore, phagocytosis was first studied at 0°C to determine the attachment of particles to the cell surface. After 30 min incubation at 0°C there was no enhancement of the attachment phase by PTU as assessed by both phase contrast microscopy and spectrophotometry (O.D. : o.oo6f 0.001, nine determinations). Table IV shows the amount of particles actually ingested after 30 min incubation a t 37°C. Propylthiouracil, uracil and thiourea had no effect on phagocytosis of latex particles by human polymorphonuclear leucocytes. The average number of particles ingested by each leucocyte was 8f 0.5 (meanf S.E.) as calculated from the extinction coefficient (2.4 x I O - ~ O.D.
f 129 (9) Experiment
2700 f 361 (9) < 0.01
Methimarole 1641 f.237 (7) 2646 f 362 (7) < 0.01
1406 f 247 (3) I943 f 199(3)
1418 f 430 (3) 1361 f 394 (3)
Adenine -1669 f 176(3) I739 &202 (3) >O.I
f265 (4) 2172
Urea 1613 f 167 (3) I750 f322 (3) > 0.1
Min-Fu Tsati and Patricia A. McIntyre
units/pg per ml at 274 nni) and the density (1.03 g/ml at 20°C)ofpolyvinyl toluene (Weisman & Korn, 1967).Lack of stimulation of phagocytosis by PTU in this experimental situation was not because the phagocytic capacity of leucocytes was already saturated after 30 min incubation with 0.2% polyvinyl toluene latex particles. There was a 50% increase in the particle uptake after incubation with 0.5% particles for 3 0 min (O.D.: 0.13ofo.013, nine determinations, P < 0.01). TABLE 111. Effect of PTU on [14C]C02release from [~-~~C]glucose by PMN leucocytes from patients with chronic granulomatous disease (CGD)* Resting cells
CGD Patient I (P.R.) Patient 2 (B.P.) Control
* The results from patients with CGD are the mean (dpm) of duplicate experiments. Those of normal control are from Table I. The radioactivity added to each flask was 2 x 105dpm. TABLE IV. Effect of propylthiouracil, thiourea and uracil on phagocytosis of latex particles by human PMN leucocytes* Conditions
P value (vs. confror)
Control +PTU +Thiourea +Uracil
0.085 f 0.009 0.092 f0.004
0.083 f 0.006 0.086k 0.010
> 0.1 > 0.1 > 0.1
* The results are expressed as mean f standard error of the mean (O.D. absorbance at 274 nm of the dioxane extracted polyvinyl toluene latex). The concentration of chemicals used was 0.1 m ~Number . of experiments: 9. Efect ofsome metabolic inhibitors on PTUstimulated HMS. It has been repeatedly shown that potassium cyanide inhibits haem-containing enzymes, has no effect on phagocytosis, but stimulates the hexose monophosphate shunt in leucocytes during phagocytosis. Iodoacetate which inhibits glycolysis, inhibits phagocytosis and phagocytosis associated HMS activity (Sbarra & Karnovsky, 1959;Reed, 1969; Reed & Tepperman, 1969).These two inhibitors were investigated in the presence and absence of PTU in cells during phagocytosis. As shown in Table V, in the absence of PTU there was a three-fold increase of C 0 2release from glucose-carbon-1 by I m~ KCN, but 0.1 m~ iodoacetate inhibited COz release by 50%.
Propylthiouracil and Polymorph Function
In contrast, in the presence of both propylthiouracil and potassium cyanide, stimulation was not significantly greater than that observed with KCN alone (P>o.I). Iodoacetate in the presence of PTU produced a 50% inhibition, but twice the amount of COz production was still observed as compared to control values with iodoacetate alone. Efect of PTU on [14C]formate oxidation. Our data indicated that PTU stimulated HMS activity in PMN during phagocytosis and that this effect was not mediated through increased capacity of phagocytosis of PTU treated cells and was insensitive to iodoacetate. Abundant evidence (Reed, 1969 ;Noseworthy& Karnovsky, 1972) indicates that HzOz,generated during phagocytosis, stimulates the HMS via the glutathione cycle. One pathway of HzO, utilization during phagocytosis is the peroxidation of added formate carried out by catalase (Iyer et a/, 1961). Therefore, the effect of PTU on [14C]formateoxidation was examined. Propylthiouracil had no effect on formate oxidation in either resting cells or cells during phagocytosis (n = 7, P>o.I). E f i c t ofexogeneous Hz0, on HMS. Since PTU stimulated HMS only in cells during phagocytosis, but had no effect on formate oxidation, it was possible that the PTU effect was dependent on H z 0 2generated during phagocytosis, rather than a further stimulation ofHzOz production. This possibility was tested by studies of the effect of H 2 0 2 on resting cells. As can be seen in Table VI, hydrogen peroxide stimulated glucose-I-C oxidation in resting PMN and a further stimulation was noted with PTU. KCN and iodoacetate had no effect on the PTU stimulated HMS activity. Potassium cyanide is a potent catalase inhibitor; it should make more HzOz available intracellularly for the stimulation of HMS. The fact that KCN did not cause further stimulation of glucose-I-C oxidation indicated that there was an adequate supply of H202 from the incubation medium under our experimental conditions. TABLE V. Effect of metabolic inhibitors on [14C]C02 release from [~-~~C]glucose by human PMN leucocytes during phagocytosis* Conditions
Without PTU Control +KCN (I m) +Iodoacetate (0.1m) With PTU (0.1 mM) Control KCN (I m) +Iodoacetate (0.1mM)
1155 f I44 43Wf 532 556f 104
5019 f 780
Efect of PTU on glucose-6-phosphate dehydrogenase (G6PD) and 6phosphogluconate dehydrogenase (6PGD) activity. Another possibility for the stimulation of HMS by PTU in cells during phagocytosis was the stimulation or activation of HMS enzymes. The activities of G6PD and 6PGD in the soluble fraction were measured after pre-incubation with and without
Min-Fu Tsan and Patricia A. McIntyre TABLEVI. Effect of H 2 0 2 on [14C]C02 release from by resting human PMN leucocytes* [~-'~C]glucose I Conditions
Control +PTU +HzO2 +H202+PTU +H20z+PTU+KCN + H 2 0 z+PTU+Iodoacetate
[ 1 * C ] C 0 2 production
314f37 373 f88
834 f49t 1402f29St 163I f 299 1 2 4 f a78
* The results are expressed as meanfstandard error of the mean (dpm) of four duplicate experiments. The concentration used: PTU: 0.1 mM, H202: 4.4 m,KCN: I m ~Iodoacetate: , 0.1 m ~ . t The differencesbetween control and H200,H202 and H202+PTU were significant (P values