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Sodium diethyl dithiocarbamate (DTC) is a low molecular weight sulfur compound which has been previously shown to have immunomodulatory properties. In the present report, the effects of DTC on the mononuclear phagocytic system were studied in guinea pigs. The colloidal gold clearance was enhanced in animals which received an i.v. injection of 10 mg DTC on day – 13, but not in animals treated on day – 8 with DTC. DTC treatment modified neither Tc sulfur colloid uptake nor activation markers in peritoneal macrophage monolayers. These results show that DTC which modulates both delayed hypersensitivity and antibody synthesis only enhances nonspecific binding of macrophages but leaves unaffected endocytosis and metabolic status of macrophages. Correspondence to: P. J. Neveu, Groupe de Recherche de Biochimie Hématologique, UER de Médecine, 34, rue du Jardin des Plantes, F–86034 Poitiers (France) Sodium diethyl dithiocarbamate (DTC) is a low molecular weight sulfur compound which has previously been chosen to exemplify the role of the thiol moiety of the levamisole molecule [1]. It was demonstrated to have immunomodulatory properties in mice [1] and in guinea pigs [2]. In guinea pigs, DTC enhanced delayed hypersensitivity reactions [2] and mitogen-induced proliferation of T and B spleen lymphocytes [3] only after a lag period suggesting that DTC, acting possibly at the macrophage level, modulates the immune responses through indirect pathways. In the present report, the effects of DTC on the mononuclear phagocytic system were studied in guinea pigs. The clearance of colloidal gold was determined according to the method described by Haugen et al. [4]. Guinea pigs were injected with 2 mg/kg body weight of colloidal radioactive gold (198Au; diameter of particles 300 Å; CEA, Gif-sur-Yvette, France). Blood samples were collected by retroorbital sinus puncture on heparinized tubes 3, 5, 10 and 15 min after gold injection. The activity of 100-μl samples was measured within an interval of 1 h, using a SAIP γ counter. The phagocytic index was calculated according to the formula: κ = logC0-logC| T1-T0 where Cj and Co represent the gold concentration at time Tl and TO (in min) expressed as cpm. Cpm were plotted in a semilogarithmic scale in relation to time. The best fitting curve for these points, called the elimination curve, was drawn and the phagocytic index calculated. In a first experiment, guinea pigs were treated i.v. with 10 mg of DTC, 6 h before or at the same time they were injected i.v. with radioactive colloidal gold. Whether guinea pigs were treated with DTC 6 h before or at the time they were injected i.v. with radioactive colloidal gold, the phagocytic index was similar to that observed in untreated controls (K was 0.12 ± 0.01 ‚ 0.13 ± 0.01 ‚ and 0.13 ± 0.02, respectively). In a second experiment, groups of 4 guinea pigs were D ow nl oa de d by : 54 .1 91 .4 0. 80 9 /1 6/ 20 17 1 1: 56 :1 8 A M treated i.v. with 10 mg of DTC 13 or 8 days before they were injected with colloidal radioactive gold (fig. 1). In guinea pigs treated with DTC 8 days before the injection of colloid, the phagocytic index was similar to that of controls, but animals treated 13 days before test exhibited an enhanced phagocytic index. An increase or a decrease of the clearance rate induced by a variety of chemical or biological substances has been interpreted as a stimulation or blockade of the reticuloendothelial system (RES) activity [5]. From these results, DTC appeared to stimulate the activity of the RES after a lag period. Effect of DTC on the Mononuclear Phagocytic System 277 Table I. Organ distribution of colloidal gold in guinea pigs treated with DTC Group Liver Spleen Lung Kidney Skin Muscle cpm/g cpm/g cpm/g cpm/g weight/g cpm/g cpm/organ cpm/organ weight/g cpm/g cpm/ cpm/organ organ cpm injected,’ cpm injected, % 19.25 1,184,000 22,797,847 96.56 ± ± 2.10 263,986 0.72 796,032 556,471 2.36 ± ± 0.05 184,000 0.62 818,450 510,303 2.16 ± ± 0.08 88,577 0.77 860,102 668,457 2.83 ± ± 0.11 148,988 6,736 2,677 2,209 1,174 ± ± ± ± 3,535 1,642 1,093 612 6,990 3,777 1,567 1,114 ± ± ± ± 2,893 1,309 1,007 540 6,220 2,047 1,157 1,281 ± ± ± ± 2,220 563 245 777 Guinea pigs were treated with an i.v. injection of 10 mg of DTC 13 days (group 1) or 6 h (group 2) before test. Guinea pigs of group 3 were untreated controls. Table II. 99mTc sulfur colloid uptake, H202 release, protein content and enzymatic activities in resident adherent peritoneal cells from guinea pigs treated with DTC D ow nl oa de d by : 54 .1 91 .4 0. 80 9 /1 6/ 20 17 1 1: 56 :1 8 A M The organ distribution of colloidal gold in animals injected i.v. with 100 μCi of the radioactivegold colloidal solution 13 days (group 1) or 6 h (group 2) after they were treated with 10 mg ofDTC was similar to that observed in controls (group 3) which received only colloidal gold (tableI). More than 90% of the injected colloidal gold was recovered in liver and spleen as previouslyreported [6]. Furthermore, the weight of liver and spleen was not increased after DTC treatment.These results, similar to those obtained using le-vamisole as immunostimulant [7], suggested thatthe effects of DTC were not due to hyperplasia or to hypertrophy of the RES but rather to anactivation of the mononuclear phagocytic system. The 99mTc sulfur colloid uptake andenzymatic markers were determined in peritoneal macrophage monolayers. The uptake of 99mTcsulfur colloid was performed as previously described [8]. Hydrogen peroxide release byperitoneal adherent cells was studied using a fluoro-metric assay described by Nathan and Root[9]. The amount of macrophage monolayer protein was determined by the method of Lowry etal. [10] using bovine serum albumin (Biomérieux, Lyon, France) as standard. Enzymaticactivities were measured in 0.1 % Triton X-100 adherent cell lysates. ß-Glucuronidase (EC3.2.1.31) activity was determined by the method of Talalay et al. [11] using phenolphthalein-/3⁄8lucuro-nide as substrate, and acid phosphatase (EC 3.1.3.2.) activity by the method describedby Andersh and Szcypinski[l2] using 4-nitrophenyl phosphate in sodium acetate buffer assubstrate. Peritoneal macrophage monolayers from groups of 5 guinea pigs treated with 10 mgDTC 14 or 6 days before test, incorporated as much 99mTc sulfur colloid as macrophages fromuntreated control animals (table II). On the other hand, macrophages from untreated and DTC-278Neveu/Vincendeau 5 1AcknowledgementsWe wish to thank Prof. G. Renoux for his critical review of the manuscript. This work wassupported by Inserm grant No. 79.3.047.2 and by the Fondation Langlois.2 3-References1 Renoux, G.; Renoux, ML: Roles of the imidazole or thiol moietyon the immunostimulantaction of levamisole; in Chirigos,’ ¦ – Control of neoplasia by modulation of the immunesystem,pp. 67–80 (Raven Press, New York 1977).Neveu, P.J.: The effects of thiol moiety of levamisoleon bothκ = 0.11 ± 0.01 cellular and humoral immunity during the early response to ahapten-carrier complex. Clin. exp. Immunol. 32: 419–422(1978).\ 3 Neveu, P.J.; Perdoux, D.;Lafleur, L.: In vivo enhancement of\ mitogen induced lymphocyte DNA synthesis by sodium dieth-\ yl-dithiocarbamate. Int. J. Immunopharmacol. 4: 9–13 (1982).κ015+001 4 Haugen, J.; Bassøe, H.H.; Flood, P.R.: Phagocytosis in rabbitstreated with oxyphenbutazone and cortisone, studied by gold clearance test and electronmicroscopy. J. reticuloendoth. Soc. 6: 184–193(1969).5 Cooper, G.N.; Stuart, A.E.: Susceptibility of mice to pneumoι coccalinfection after modification of the reticuloendothelial3 5 10Fig. 1. Kinetics of phagocytosis of colloidal gold ( phagocytic indices (K values) in untreatedguinea pigs (– in guinea pigs treated i.v. with 10 mg of DTC on day -8 (---) or on day-13 (–). Downloadedby: 54.191.40.80-9/16/201711:56:18AM 15 mm system with simple lipids. J. Path. Bact. 83: 227–243(1962).Au) and )andtreated guinea pigs did not spontaneously release significant amounts of hydrogen peroxide. Inthe presence of PMA (12–0-tetradecanoyl-phorbol-13-acetate; Sigma, St. Louis, Mo.),macrophages from DTC-treated animals released as little 3⁄4C1⁄8 as macrophages from controlanimals. Protein content as well as acid phosphatase and ß-glucuronidase activities were similarin macrophages from DTC-treated and from untreated control animals. Direct effects of DTC onthe mononuclear phagocytic cells resulting in their activation could be excluded.From the results reported here, it could be speculated that in DTC-treated guinea pigs, themembrane of macrophages is altered so that nonspecific binding is enhanced whereasendocytosis and metabolic status of macrophages are unchanged. This can explain the greaternumber of Listeria monocytogenes forming colonies in spleen and liver of DTC-treated ascompared to control animals [13]. The trapping of listeria is enhanced while phagocytic activityis not. The mechanisms by which DTC can enhance the nonspecific binding of macrophages areunder investigation.Zilversmit, D.B.; Boyd, G.A.; Brucer, M.: The effect of particle size on blood clearance andtissue distribution of radioactive gold colloids. J. Lab. clin. Med. 40: 255–620 (1952).Hoebecke, J.; Franchi, G.: Influence of tetramisole and its optical isomers on the mononuclearphagocytic system. Effect on carbon clearance in mice. J. reticuloendoth. Soc. 14: 317–322(1973).Vincendeau, P.; Vincendeau-Scherrer, C; Ducassou, D.; Bez-ian, J.H.: Uptake of 99mTc-sulphurcolloid by mononuclear phagocytes. Int. J.nucl. Med. Biol. 7; 383–385(1980).Nathan, C.F.; Root, R.K.: Hydrogen peroxide release from mouse peritoneal macrophages.Dependence on sequential activation and triggering. J. exp. Med. 146: 1648–1662(1977). Lowry, O.H.; Rosebrough, N.J.; Farr, A.L.; Randall, R.J.: Protein measurement with the Folinphenol reagent. J. biol. Chem. 193: 265–275(1951).Talalay, P.; Fishman, W.H.; Huggins, G.: Chromogenic substrates. II. Phenolphthaleinglucuronic acid as substrate for the assay of glucuronidase activity. J. biol. Chem. 166: 757–768(1946).Andersh, M.A.; Szcypinski, A.J.: Use of / > -nitrophenyl phosphate as the substrate indetermination of serum acid phosphatase. Am. J. clin. Path. 17: 571–577(1947).Neveu, P.J.; Lagrange, P.H.; Thierry, D.; Perdoux, D.: Diethyl-dithiocarbamate de sodium etsystème réticulo-endothélial. Nouv. Revue fr. Hémat. 22: C139 (1980). Downloadedby: 54.191.40.80-9/16/201711:56:18AM