were opsonized with freshautologous serum as fol- lows. The zymosan was suspended in normal saline at a concentration of 50 mg/ml. One part of the zymosan ...
Vol. 43, No. 3
INFECTION AND IMMUNITY, Mar. 1984, p. 846-849 0019-9567/84/030846-04$02.00/0 Copyright © 1984, American Society for Microbiology
Extracellular Metabolism of Thyroid Hormones by Stimulated Granulocytes MOHAN K. RAO AND ARTHUR L. SAGONE, JR.* Division of Hematology and Oncology, The Ohio State University College of Medicine, Columbus, Ohio 43210 Received 22 August 1983/Accepted 28 November 1983
Stimulated polymorphonuclear leukocytes have been shown by this laboratory to release a reactiveoxygen species (ROS) which is detectable in the supernatant and is capable of oxidizing reduced glutathione and reacting with methionine (Sagone et al., Blood 63:96-104, 1984). This ROS is dependent on H2O0 and heme for its production and is postulated to be a stable oxidant derived from hypochlorous acid, such as a chloramine. Further, this ROS was also shown to be able to oxidize and fix iodide to protein. This latter characteristic was the theoretical basis for our present study in which the same ROS was shown to be able to carry out the iodination of 3,3,5'-triiodothyronine to thyroxine in the presence of I-. Our results provide further support that granulocytes have a role in the peripheral utilization of thyroid hormones in patients with infectious diseases or other illnesses in which granulocytes may be activated, and our results indicate that the reactions may occur extracellularly. PMNs in vivo. In addition these compounds may be important mediators of inflammation (12a). The chemical characteristics of this stable oxidant, combined with observations by others indicating that the granulocytes metabolize thyroid hormone (3, 5, 10, 19), led to our current experiments. Several investigators have reported evidence that stimulated granulocytes have the capacity to deiodinate thyroid hormones (10, 19) and that this property of leukocytes may relate to the increased turnover of thyroid hormones observed in patients with infections (3, 5). Further, Klebanoff and Green suggested that the metabolism of thyroid hormones by PMNs may be in part related to a ROS produced by the MPO enzyme system (10). Therefore, the capacity of the stable oxidant released by PMNs to oxidize I to 12 and to fix halogen to protein suggested to us that it might also be able to react with thyroid hormones. To establish that the oxidant released by PMNs could metabolize thyroid hormones, we studied the capacity of activated supernatants from stimulated granulocytes to convert 3,3,5'triiodothyronine (T3) to thyroxine (T4) in the presence of halogen. This system was chosen because a sensitive immunoassay is available for the measurement of T4. We believed that these experiments would provide further evidence for the iodine-fixing capacity of the stable ROS released by PMNs, as well as suggest a role for this oxidant in the metabolism of thyroid hormones in diseases in which granulocytes are activated. We have shown that the stable oxidant released by granulocytes is able to produce T4 when incubated with T3 and potassium iodide.
Stimulated polymorphonuclear leukocytes (PMNs) generate a number of reactive-oxygen species (ROS). These include superoxide (02-), hydrogen peroxide (H202), and hydroxyl radical (OH .). In addition, hypochlorous acid is produced after the reduction of H202 by the myeloperoxidase (MPO) system. The ROS generated by this latter system may be important in several reactions known to be mediated by PMNs. These include the capacity of activated granulocyte suspensions to fix halogen onto protein (6, 7, 9, 12) and to oxidize reduced soluble sulfhydryls (11), methionine (16), and N-formylmethionyl proteins (17), as well as to inactivate methionine enkephalin (18) and serum alpha protease inhibitors (1). We have recently demonstrated that stimulated granulocytes release a ROS which is stable enough to be detected in the supernatant (12a). This compound is dependent on H202 and heme for its production and is most likely a chloramine (12a). The following equations indicate the probable mechanism for the production of this oxidant: HOCl H202 heme or (hypochlorous acid) (hydrogen peroxide)
myeloperoxidase HOCl + R NH2 (amino acid)
R NH Cl (n-chloramine)
Once formed, this stable oxidant is able to oxidize sulfhydryls, oxidize I to 12, and halogenate proteins. Further, the stable oxidant described in our experiments may be the same ROS mediating the kinds of reactions described above at other laboratories. Thomas has demonstrated that the chloramines derived from HOCI are bactericidal (14, 15). Therefore, our recent studies demonstrating that PMNs have the capacity to generate these compounds suggest that chloramines may be important in the bactericidal capacity of
*
MATERIALS AND METHODS Cell preparation. Venous blood from healthy volunteers was collected in EDTA (0.3 ml of 5% solution per 10 ml of whole blood. Leukocytes were isolated by dextran sedimentation. Whole blood was incubated at 37°C for 1 h in 0.1 volume of 4% dextran. The plasma, containing leukocytes and platelets, was removed, mixed in Seligman balanced salt solution, and layered over a standard Ficoll-Hypaque mixture to remove platelets and mononuclear cells (11, 13). This mixture was centrifuged for 35 min at 800 x g and then aspirated down to the bottom which contained granulocytes
Corresponding author. 846
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and a few erythrocytes Hypotonic-shock lysis eliminated the
remaining erythrocytes. Cells were washed with Seligman balanced salt solution and suspended in Dulbecco phosphate-buffered saline containing 100 mg/dl of glucose. The final preparation contained more than 99% granulocytes and less than 1% mononuclear cells. Cells were counted electronically with a model FN Coulter Counter. Conversion of T3 to T4 by supernatants of stimulated granulocytes. A suspension containing 15 x 106 cells was placed in Nalgene tubes (17 by 100 mm) with 7-mm magnetic stir bars. Zymosan particles (Sigma Chemical Co., St. Louis, Mo.) were opsonized with fresh autologous serum as follows. The zymosan was suspended in normal saline at a concentration of 50 mg/ml. One part of the zymosan suspension was incubated with three parts of fresh autologous serum at 37°C for 30 min. The suspension was then centrifuged, and most of the supernatant was removed. The zymosan was suspended in buffer at a concentration of ca. 12.5 mg/ml. After a 10-min preincubation at 37°C, zymosan (5 mg per 20 x 106 PMNs) was added to the above tubes. An identical amount of buffer was added to control tubes. The volume was adjusted with medium so that the final concentration of cells was 10 x 106 to 11 x 106 cells per ml. This concentration was maintained for all of the experiments discussed in this study. The samples were then incubated for 15 min at 37°C. After this, the tubes were placed in an ice bath for 5 min, and then spun for 20 min at 40C at 27,700 x g on a Sorvall RC-2B centrifuge. One milliliter of supernatant was drawn off from each tube and combined with 0.2 ml of a 31.25 mM KI solution (giving a 5 mM KI final concentration) and 0.05 ml of a 25-,ug/ml solution of T3 (giving a 1-,ug/ml final concentration). The solutions were then incubated for 15 min at 37°C. Samples were assayed for T4 with a Tetra Tab RIA T4 Diagnostic Kit obtained from Nuclear Medical Laboratories Inc., Dallas, Tex. A concentrated stock T3 solution was made up in 5 ml of distilled water by dissolving T3 in 1 N NaOH and titrating to pH 10 to 11. The diluted T3 solution added to supernatants had a pH of between 9 and 10. For experiments in which scavengers and inhibitors were added to the granulocyte suspensions, the tubes had 18 x 106 cells per 1.73-ml total volume. A 0.05-ml volume of scavengers and inhibitors (0.225 mg of azide per ml, 0.865 mg of catalase per ml, 0.051 ml of 1.37 M mannitol, 99.72 mg of benzoate per ml, 20 mM reduced glutathione [GSH], 1.03 mg of methionine per ml, 0.346 mg of superoxide dismutase [SOD] per ml, 0.170 mg of sodium cyanide per ml) was added to the cell suspensions before the 10-min preincubation. The procedure for obtaining the supernatants and reacting them with T3 and KI was as described above. For experiments in which scavengers and inhibitors were added to the supernatants, the tubes had 18 x 106 cells per 1.68-ml total volume. A 0.05-ml volume of scavengers or inhibitors (0.169 mg of azide per ml, 0.65 mg catalase per ml, 0.778 mg of methionine per ml, 2 mM GSH) was added to the 1-ml supernatant. A 0.05-ml volume of T3 (25 jig/ml) and 0.2 ml of 31.25 mM KI were then added as soon as possible. The samples were incubated for 15 min at 37°C. To terminate the reaction, we froze the samples in a -80°C freezer where they were kept until they were to be assayed. Reagents. Glutathione, SOD (type 1; 2,500 U/mg of protein), bovine liver catalase (16,000 U/mg), sodium azide, sodium benzoate, methionine, 3,3',5-triiodo-L-thyronine (free acid), and zymosan were obtained from Sigma Chemical Co. Mannitol was obtained from Abbott Laboratories, North Chicago, Ill. Sodium cynanide was obtained from Fisher Scientific Co., Pittsburgh, Pa. The T4 assay was
CONVERSION OF T3 TO T4 BY GRANULOCYTES
847
performed using the Tetra Tab RIA T4 Diagnostic Kit from Nuclear Medical Laboratories, Inc. Statistical analysis. The data were analyzed according to tests for dependent or independent samples. Results are expressed as the means ± standard deviations. RESULTS Effect of the addition of T3 and KI to supernatants of stimulated granulocytes. The addition of T3 (final concentration of 1 jig/ml) and 5 mM KI (molar excess) to 1 ml of supernatant from granulocytes stimulated by opsonized zymosan particles resulted in the formation of T4 (Table 1). In one experiment in which a serial dilution of the number of cells employed was used, there was a progressive decline in the amount of T4 produced (data not shown). A significant difference exists between the T4 production of stimulated and unstimulated granulocytes. When no T3 was added, the T4 assay yielded a value which was below the normal background (see the value obtained for "media alone + T3 or medium + zymosan + T3") for this assay. We also used unopsonized zymosan to stimulate the granulocytes. This resulted in T4 production similar to that of unstimulated granulocytes (Table 1). Addition of metabolic scavengers and inhibitors to the granulocyte suspensions. The granulocyte suspensions were supplemented with given reagents. After incubation with zymosan, 1 ml of supernatants was recovered in the usual manner, and the capacity of the supernatant to fix 1- to T3 was determined (Table 2). The scavengers benzoate, mannitol, and SOD, when added to the granulocyte suspension, had no effect on the production of the factor, as evidenced by T4 amounts similar to those of the controls. The addition of azide and catalase diminished the production of T4. The presence of GSH and methionine in the granulocyte suspensions reduced the amount of T4 formed. Phenol also had an inhibitory effect (data not shown). Addition of metabolic inhibitors and scavengers to the supernatants. In another set of experiments, the reagents were added to the 1 ml of supernatants along with the KI and T3, and the mixture was incubated for 15 min and assayed for TABLE 1. Effect of supernatants of stimulated granulocytes when exposed to T3 and KIa Type of supernatant
No. of experi-
T4 (,ug/dl)
P value
ments
18 7.0 ± 2.1 Stimulated (control) 2.4 + 0.1 18 < 0.001 Unstimulated 2 1.0 Stimulated; no T3 added 2 2.56 Stimulated with unopsonized zymosan 2.4 ± 0.5 21 < 0.001 Media alone + T3 or media + zymosan + T3 a For these experiments, 1 ml of supernatants from the zymosanstimulated granulocytes (18 x 10"/1.68 ml) was drawn off from the appropriate tubes. The supernatant was mixed with 0.05 ml of T3 (25 ,ug/ml) and 0.2 ml of KI (31.25 mM), incubated for 15 min at 37°C, and assayed for T4. Samples which demonstrated the normal background for the T4 assay were those in which supernatants from the tubes containing media plus zymosan and those containing media alone were combined with T3 and KI. The amount of KI present was 5 mM, and the T3 concentration was 1 ,ug/ml. The T4 value for stimulated cells served as the control against which all P values given here and on other tables are referenced. b The value was derived from two experiments. In both cases, the value was 2.5.
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TABLE 2. Effect of the addition of metabolic scavengers and inhibitors to granulocyte suspensionsa No. of
Supplement
experi-
T4 (p.g/dl)
P value
ments
7.0 ± 2.1 18 Stimulated (control)
