Pergamon Free Radical Biology & Medicine, Vol. 19, No. 3, pp. 359-363, 1995

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Brief Communication

O N T H E M E C H A N I S M O F T H Y R O I D H O R M O N E - I N D U C E D

R E S P I R A T O R Y B U R S T A C T I V I T Y IN R A T

P O L Y M O R P H O N U C L E A R L E U K O C Y T E S

VIRGINIA FERNANDEZ and LUIS A. VIDELA

Department of Biochemistry, Faculty of Medicine, University of Chile, Santiago, Chile

(Received 19 July 1994; Revised 16 December 1994; Accepted 20 December 1994)

Abstract--Administrat ion of single doses of 0. i mg L-triiodothyronine (T3)/kg for 3 consecutive days to fed rats produced a drastic increase in the respiratory burst activity of isolated polymorphonuclear leukocytes (PMN), stimulated with semm- opsonized zymosan. This effect was evidenced by the 3.8-fold increment in the integrated chemiluminescence, and seems to be primarily related to the enhanced activity of NADPH oxidase elicited by T3 treatment, with the observed higher myeloperoxidase activity playing a contributory role. In these conditions, hyperthyroidism determines a net enhancement in the oxidant capacity of PMN, as the increased rate of O[- generation found occurs in the absence of changes in the activity of superoxide dismutase.

Keywords-42hemiluminescence, Polymorphonuclear leukocytes, Respiratory burst activity, Superoxide radical production, Superoxide dismutase, Myeloperoxidase, NADPH oxidase, Hyperthyroidism, Free radicals

INTRODUCTION

Thyroid calorigenesis has been suggested to be primar- ily determined by the interaction of thyroid hormones with nuclear receptors of target cells.~ This interaction leads to an adaptive enhancement in microsomal, 2 mi- tochondrial, 3 and peroxisomal 4 mass, as well as in the activity of enzymes and in the content of biomolccules related to the intermediary metabolism and electron transport systems/ As a consequence, thyroid hor- mone-induced acceleration of the energy metabolism 6 determines increased rates of O[- and/or H202 produc- tion at the microsomal, 7 mitochondrial, 8 and peroxi- somal8 sites, as demonstrated for the liver cell.

Apart from the hcpatocyte, PMN have been shown to have saturable nuclear binding sites for T3, 9 display a calorigenic response in thyrotoxic patients, to and manifest elevated rates of oxidative metabolism upon stimulation, when assayed in whole blood samples either from rats given 3 consecutive daily doses of 0.1 mg T 3 / k g or from hyperthyroid patients." This was evidenced by the mark- edly increased luminol-amplified CL after stimulation by zymosan, an effect that is characterized by being (a) pro-

Address correspondence to: Virginia Femfindez, Departamento de Bioqulmica, Facultad de Medicina, Universidad de Chile, Casilla 70086, Santiago-7, Chile.

duced independently of the opsonization of the zymosan particles, (b) drastically inhibited by azide added in vitro, and (c) reduced to control values by propylthiouracil treat- ment (hyperthyroid patients given 400 mg/day for 2-3 months). II Although thyroid hormones are suitable for free radical reactions due to their phenolic structures, addi- tion of T3 and/or thyroxine at picomolar concentrations to whole blood samples from euthyroid rats failed to produce significant changes in zymosan-induced light emission, when compared to values obtained in their absence. ~1 Based on these findings, it was suggested that hyperthy- roidism-induced respiratory burst activity of whole blood phagocytes is related to adaptive changes in the intraceUu- lar mechanisms involved, rather than changes in the op- sonic capacity of plasma or direct actions of the thyroid hormone molecule." In order to elucidate the mechanisms underlying this action of thyroid hormone on phagocyte oxidative metabolism, the present work assesses the influ- ence of T3 treatment on the major components of the respiratory burst in isolated rat PMN, namely, NADPH oxidase and MPO activities, in relation to the capacity of the cells to generate O~- and emit light.

MATERIALS AND METHODS

Animals

Female Sprague Dawley rats (Instituto de Salud Pdblica, Santiago, Chile) weighing 350-400 g were

359

360 V. FERNANDEZ and L. A. VIDELA

fed ad libitum and received daily intraperitoneal injec- tions of either T3 (0.1 mg/kg for 3 consecutive days) or equivalent volumes of T3 diluent (0.1 N NaOH) (controls). ~l After treatment, s e r u m T 3 levels mea- sured by the Gamma Coat TM [ 125I]T3 Radioimmunoas- say Kit (Baxter Healthcare Corp., Cambridge, MA) were significantly increased (controls, 75 ___ 8 ng/dl [n = 10]; T3-treated rats, 365 ___ 43 [n = 10]; p < 0.001 ). In these conditions~ the rectal temperature of hyperthyroid rats (measured with a thermocoupla Cole-Parmer model 8112-20, Cole-Parmer Instru- ment Co., Chicago, IL) was significantly enhanced compared to controls (controls, 37.7 + 0.1°C [n = 16] ; T3-treated rats, 38.4 + 0.1 [n = 19] ; p < 0.0005), suggesting a T~-induced calorigenic state.

Isolation of PMN

Blood samples from experimental animals were ob- tained by cardiac puncture using heparinized plastic syringes and were processed immediately. Portions of 4 ml of blood were mixed with 0.7 ml of 6% dextran prepared in 0.9% NaCI, and the erythrocytes were al- lowed to settle down for 30 min. The plasma was removed and layered on 3 ml of a Ficol l-Hypaque solution (density = 1.077 g/ml) and centrifuged at 400 x g for 30 min. The supernatant, containing lym- phocytes and monocytes in the plasma-Ficoll in- terphase, was discarded. The pellet containing PMN and residual erythrocytes was suspended by gentle mixing in 4 ml of 0.83% NH4C1, and settled for 10 rain in order to lyse erythrocytes. After centrifugation at 400 X g for 10 min, the pellet was washed twice with 4 ml of PBS 12 pH 7.0, and the final pellet was suspended in appropriate volumes (0.05-0.15 ml) of PBS. All procedures were carried out at room tempera- ture using polypropylene tubes and pipettes, giving a greater than 95% purity and a viability of 82 ___ 1% (n = 58), assessed by the trypan blue exclusion method. Biochemical parameters measured in whole cells were performed within 3 h after cardiac puncture.

Biochemical parameters

Visible CL was measured in a Beckman LS- 6000TA liquid scintillation spectrometer, using single photon monitoring (Beckman Instruments Corp., Ful- lerton, CA) at 25°C. Measurements were carried out in a reaction medium (final volume of 2.0 ml) containing CVB, 12 0.05 ml of 0.8 mM luminol, and 0.02 ml of PMN suspension (1.0 to 1.5 x 10 6 cells/ml). The reaction was started by the addition of 0.05 ml of 2.5 mg/ml serum-opsonized zymosan prepared as de- scribed by Allen et al. ~2 (time zero), and CL was

recorded at 2 min intervals until peak values were observed. Backgrounds consisting of CVB alone or in the presence of either luminol, PMN suspensions, zymosan, or luminol plus zymosan did not emit light. Results are expressed either as counts/min/viable cell or integrated CL (area under the time curves of counts/ min/viable cell between 0 - 3 2 rain), in counts/cell. NADPH oxidase was measured by recording the oxida- tion of NADPH at 340 nm and 28°C in particulate preparations obtained from 4 - 8 x 10 6 viable cells/ml stimulated with serum-opsonized zymosan, ~2 as de- scribed by Tauber.~3 The results are expressed as nmol of oxidized NADPH/min/mg protein, using the extinc- tion coefficient of 6.22 mM -l x cm -~ . The protein content of the particulate preparations was measured according to Lowry et al. 14 02- generation was as- sessed by the reduction of ferricytochrome c at 550 nm and 37°C, after stimulation of 105-106 cells/ml with opsonized zymosan, 12 as described by Cohen/5 The results are expressed as nmol/min/106 viable cells, using the extinction coefficient of 0.0211 nM -j x cm -l . MPO activity was measured at 655 nm and 37°C using tetramethyl benzidine and H202, after re- leasing the enzyme from 1-2 x 10 6 cells/ml with cytochalasin B and N-formyl-methionyl-leucyl-phe- nylalanine, as described by Suzuki et al. 16 The results are expressed as AA655/min/106 viable cells. SOD activity was assessed by the inhibition of the reduction of nitroblue tetrazolium (NBT) at 560 nm and 25°C, using the xanthine/xanthine oxidase system to gener- ate O~- and 1 X 10 6 homogenized cells/ml, according to Oberley and Spitz. 17 The results are expressed as Units/106 cells (One Unit of SOD activity corresponds to the amount of protein giving half-maximal inhibition of NBT reduction). Total GSH equivalents (GSH + 2GSSG) were determined by the catalytic assay de- scribed by Tietze, 18 at 412 nm and 30°C. Measure- ments were carried out in neutralized supernatants ob- tained after homogenization of 4 x 10 6 PMN in 1 N HCIO4 and centrifugation at 2500 x g for 10 min at 4°C. The results are expressed as nmol GSH/10 6 cells.

All reagents used were obtained from Sigma Chemi- cal Co. (St. Louis, MO). Values shown correspond to the means + SE for the indicated number of separate observations. The significance of the differences be- tween mean values was assessed by Student's t-test for unpaired data.

RESULTS AND DISCUSSION

Thyroid hormone-induced calorigenesis in the rat led to a drastic enhancement in the respiratory burst activity of isolated PMN upon stimulation (Fig. IA),

Respiratory burst of phagocytes in hyperthyroidism 361

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T3 Time (rain)

Fig. 1. Effect of the in vivo T3 administration (daily doses of 0.1 mg/kg for 3 consecutive days) on the opsonized zymosan- induced luminoi-amplified chemiluminescence of isolated rat polymorphonuclear leukocytes (PMN). Light emission is expressed either as counts/min/viable PMN (A) or as integrated chemiluminescence (0-32 min) in counts/viable PMN (B). Values shown represent the means _+ SE for seven control rats and five T3-treated animals.

as previous ly demons t ra ted in studies using whole b lood phagocytes . 1~ This effect is ev idenced by the significant 3 .8-fold increment in the luminol -ampl i f ied integrated C L of P M N from T3-treated rats, compared to that observed in P M N from control animals (Fig . 1B). Since luminol -ampl i f ied CL is known to result f rom the interact ion o f the chemi lumin igen ic probe with several oxidant species re la ted to the respira tory burst o f phagocyt ic cells, ~9 the assessment o f changes in the act ivi ty o f the enzymes invo lved in the process is required to unders tand the mechan i sms under ly ing the observed C L response e l ic i ted by T3 treatment.

Data presented in Table 1 show that T3 t reatment p roduced a signif icant increase in the N A D P H oxidase act ivi ty o f P M N over control values, e lect ron t ransport sys tem known to ca ta lyze the N A D P H - d e p e n d e n t re-

duct ion o f 02 to 0 2 - , and H202. 2° This f inding is in agreement with the drast ic enhancement in the capaci ty o f P M N from Ta-treated animals to generate O [ - (Ta- ble 1 ), one o f the oxidant species involved in the C L response associa ted with the respi ra tory burst. 12"19 In these condi t ions , increased rates o f 0 2 - product ion within the phagocyt ic vacuole o f P M N after T3 treat- ment are l ike ly to resul t in h igher rates of H202 forma-

Table 1. Biochemical Parameters Associated With the Respiratory Burst Activity of Polymorphonuclear Leukocytes Isolated From

Control Rats and T~-Treated Animals ~

Control T3-Treated Change Parameters Rats Rats (%) p

NADPH oxidase (nmol/min/ 39.0 _+ 4.5 mg protein) 23.0 _+ 2.3 (4) (4) 70 0.05

O2- generation (nmol/mird 1.30 _+ 0.23 106 cells) 0.52 _+ 0.06 (7) (8) 150 0.01

Myeloperoxidase (AA/min/106 4.20 +_ 0.43 cells) 2.37 _+ 0.06 (5) (6) 77 0.005

Superoxide dismutase 5.27 _+ 1.07 (U/106 cells) 5.14 _+ 1.03 (6) (8) 2 NS h

GSH content (nmol/106 0.47 +_ 0.04 cells) 0.46 _ 0.07 (8) (6) 2 NS

Animals were given either T3 (daily single doses of 0.1 mg/kg for 3 consecutive days) or equivalent volumes of T3 diluent (0.1 N NaOH)(controis). Values shown correspond to means _+ SE, for the number of separate experiments indicated in parentheses.

b NS, effect of T3 treatment not significantly different compared to control values.

362 V. FERNANDEZ and L. A. VIDELA

tion by spontaneous dismutat ion. This oxidant species can be used to oxid ize chlor ide to hypochlorous acid and chloramines by MPO, one o f the major granule components re leased into the phagocyt ic vacuole of PMN, 2° which was enhanced by 77% by T 3 t reatment compared to control values (Tab le 1 ). These data sug- gest that T3-induced enhancement in the act ivi ty o f N A D P H oxidase could const i tute the pr imary effect responsible for the increased respira tory burst act iv i ty e l ic i ted by thyroid hormone. In addit ion, the enhanced M P O act ivi ty may contr ibute to the respira tory burst act ivi ty induced by T3 t reatment by dr iv ing the d ismu-

tation o f O 2- into H202, when coupled to the increased act ivi ty of the N A D P H oxidase system. The M P O - H:O2-hal ide react ion represents a crucial step in the respira tory burst o f P M N under normal condi t ions, conver t ing most of the H202 produced to hypochlorous acid, 21 required for the op t imum microbic ida l act ivi ty

o f neu t roph i l s ) 2 It is impor tant to note that the increase in N A D P H oxidase and M P O activi t ies by T 3 t reatment is observed in the absence o f changes ei ther in S O D act ivi ty or in the content o f G S H of P M N (Tab le 1 ). Thus, hyper thyro id i sm determines a net increment in the prooxidant capaci ty of rat PMN, as ev idenced by the signif icant increase in the 0 2 g e n e r a t i o n / S O D ac- t ivi ty ratio (control rats, 0.12 _ 0.02 nmol O [ - / m i n / U S O D [n = 6 ] ; T3-treated rats, 0.25 ± 0.06 [n = 8] ; 108% increase; p < 0 .025) .

Al though the increased oxida t ive capaci ty induced by T 3 adminis t ra t ion on s t imula ted P M N seems to be media ted by increments in N A D P H oxidase and M P O activi t ies (Tab le 1), the involvement o f nitric oxide synthase in the observed CL response cannot be dis- carded. In fact, ac t ivated P M N have been shown to produce nitric oxide, 23.24 which, at phys io log ica l condi-

tions, can react with O [ - to generate peroxyni t r i te , a s t ronger oxidant species. 25 This aspect is current ly un-

der s tudy in our laboratory.

Acknowledgements - - This work was supported by grant 1940312 from FONDECYT. The authors thank Carmen Almeyda and Manuel Sufirez for excellent technical assistance.

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Respiratory burst of phagocytes in hyperthyroidism 363

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ABBREVIATIONS

CL - - chemiluminescence CVB--complete veronal buffer

H20/--hydrogen peroxide MPO-- myeloperoxidase PBS--phosphate buffered saline PMN-- polymorphonuclear leukocytes GSH--reduced glutathione SOD-- superoxide dismutase O2---superoxide radical T3--L-3,3 ',5-triiodothyronine