The aim of the paper was to analyze the effect of various concentrations of ascorbic acid on spontaneous apoptosis of lymphocytes.About 58% of all thymocytes are subjected to a spontaneous apoptosis, after 24-hour cultivation in the complete medium. The number of apoptotic thymocytes was much lower in the culture with different concentrations of ascorbic acid. The most dramatic effect was detected in the culture with the highest concentration of ascorbic acid (10000 μg/ml). In this culture, the number of apoptotic thymocytes was about 32%. These results, compared with the results of spontaneous apoptosis, show that the most significant inhibition of apoptosis was detected in the culture with 10000 μg/ml of ascorbic acid. High concentration of ascorbic acid can inhibit spontaneous apoptosis of thymocytes. Such inhibition of T cell apoptosis, as the effector cells in immune system, can represent one of the major factors by which ascorbic acid influence the immune system

Goran Ranković

Nenad Stojiljković

Snežana Cekić

Vladmila Bojanić

Voja Pavlović

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Acta Medica Medianae 2005. Vol. 4  Ascorbic acid modulates spontaneous thymocyte apoptosis21Institute of Physiology, Faculty of Medicine, Nis1 .Institute of Pathophysiology, Faculty of Medicine, Nis2 . .Correspondence to: Voja Pavlovic .Institute of Phisiology, Faculty of Medicine  .81 Dr Zoran Djindjic Street .18000 Niš, Serbia and Montenegro .Phone: 018/334-221 .E-mail: vojapav@yahoo.comIntroductionThymus  is  the  central  organ  of  the  immune system. The proliferation, differentiation and T-lym-phocytes selection processes take place in this organ (1). With humans, thymus reaches its maximum size during the 1st year after which its involution, as one of the examples of physiological involutions, starts (2). Thymocytes make the greatest thymus cell population. About 80% of thymocytes are located in the cortex and about 20% of thymocytes are located in the thymus medulla. All thymocytes are subjected to the process of positive or negative selection during the matura-tion process. This thymocytes selection is based on the thymocytes ability or inability to recognize antigenic peptide/own  MHC  molecule  complex.  The  negative thymocytes selection represents acquired tolerance to own antigens. It is acquired by the thymocytes’ apop-tosis or by the anergy, that is the clone inactivation (1). Apoptosis or the programmed cell death is a physio-logical process necessary for the normal development and homeostasis of an organism. The process intensity is strictly controlled (3). Increased or decreased inten-sity of this process leads to many disorders. Ascorbic  acid  is  one  of  the  basic  nutritive factors, often connected with the immune system mo-dulation. It has been proved earlier that the application of the ascorbic acid may increase T-cell proliferation and cytokines production. It also has antioxidant and anticancerogenic effect (4,5,6). All these effects point to the modulation of the nonspecific protection. The precise mechanisms for the ascorbic acid performance, aimed at the improvement of the specific protection, have not been fully examined. It is considered that one of the effects of the ascorbic acid is the modulation of T-cells survival, i.e. the modulation of the apoptosis intensity.AimsThe aim of the research is to examine the effects that different concentrations of ascorbic acid have on the intensity of the thymocyte spontaneous apoptosis.Material and MethodsWistar rats of both sexes (male and female), 9-11 weeks old were used in the experiments. The animals were bred under the standard laboratory conditions in the vivarium of the Medical Research Institute, at the Faculty of Medicine in Nis.The  animals  were  sacrificed  by  ether  inhala-tion,  administering  diethyl  ether.    Thymus  was  ex-tracted by sterile instruments and put into Petri dish with RPMI/10% FCS (Sigma, USA). After the adjacent tissue had been removed, thymus was homogenized through sterile steel net. Thymus cell suspension was resuspended and rinsed in RPMI/10% FCS. The num-ber of viable thymocytes was determined by counting in 0.1% Tripan blue solution.AScORBIc AcID MODULATES SPONTANEOUS THYMOcYTE APOPTOSISVoja Pavlovic1, Snezana Cekic1, Vladmila Bojanic2, Nenad Stojiljkovic1 and Goran Rankovic1The aim of the paper was to analyze the effect of various concentrations of ascorbic acid on spontaneous apoptosis of lymphocytes. About 58% of all thymocytes are subjected to a spontaneous apoptosis, after 24-hour cultivation in the complete medium. The number of apoptotic thymocytes was much lower in the culture with different concentrations of ascorbic acid. The most dramatic effect was detec-ted in the culture with the highest concentration of ascorbic acid (10000 µg/ml). In this culture, the number of apoptotic thymocytes was about 32%. These results, compared with the results of spontaneous apoptosis, show that the most significant inhibition of apoptosis was detected in the culture with 10000 µg/ml of ascorbic acid.High concentration of ascorbic acid can inhibit spontaneous apoptosis of thymocytes. Such inhibition of T cell apoptosis, as the effector cells in immune system, can represent one of the major factors by which ascorbic acid influence the immune system.  Acta Medica Medianae 2005;44(4): 21 – 23.Key words: ascorbic acid, thymus, apoptosiswww.medfak.ni.ac.yu/ammAscorbic acid modulates spontaneous thymocyte apoptosis  Voja Pavlović, Snežana Cekić, Vladmila Bojanić...22The obtained thymocytes were distributed in the cell-cultivating basal panels (1x106 thymocytes/200 µl) with 96 places (Sarstedt, Newton, USA) and cultivated with different concentrations of ascorbic acid (10000, 1000, 100, 10 µg/ml) (Galenika, Belgrade). The final volume of each pool was 200 µl. The cells were cul-tivated for 24 hours in a thermometer at 37ºC and 5% CO2 (Assab, Sweden).Türk’s solution for leukocyte counting was used for the morphological detection of thymocyte apopto-sis. This reagent enables the fixation and determination of the nucleus. In doing so, it makes possible a clear differentiation of the normal chromatine in viable cel-ls, as well as the chromatine condensation and karyor-rhexis of the apoptotic cells. All cultures were grown in triplicate. The obtained values are shown as mean values ± SD.Student’s T-test was used for the comparison of the obtained results.ResultsThe first part of our research was related to the examination of the intensity of the spontaneous thy-mocyte apoptosis. Actually, these results indicate that more than a half of the isolated thymocytes (appro-ximately 58%) was liable to the spontaneous apopto-sis after 24- hour cultivation in the complete medium (Graph 1). However, cultivating of the isolated thy-mocytes with different concentrations of vitamin C de-monstrate that the number of the apoptotic thymocytes decreases, proportionally to the increasing of the ascor-bic acid concentration. The most dramatic effect was detected in the cultures with the highest concentration of vitamin C (10000 µg/ml), where the percentage of the apoptotic thymocytes  decreased to approximately 32%. Also, the vitamin C concentration of 1000 µg/ml reduced the number of apoptotic lymphocytes to 48%, while the remaining concentrations of the ascorbic acid, in a much lower degree,  reduced the total number of the apoptotic cells (53-57%) in relation to the sponta-neous thymocyte apoptosis. A comparison of the obtai-ned data with the values of the spontaneous thymocyte apoptosis showed that statistically the most significant apoptosis inhibition was verified in cultures with 10000 µg/ml of vitamin C, as well as in other cultures (1000 µg/ml p < 0.01; 100 µg/ml p < 0.05), while in the cultu-re with 10 µg/ml of vitamin C, statistically significant apoptosis inhibition was not detected (Graph 2).DiscussionAscorbic acid represents a significant nutritive antioxidant. Actually, the intensity of cell and tissue damage is significantly reduced by the oxidation of the ascorbic acid into ascorbat (by tying free oxygen to it-self) (7). Nowadays, there are various data about the ascorbic acid effect on the immune system. Some au-thors deny its stimulatory effect (8, 9). The other group of authors emphasizes the protective and stimulatory effect on the immune system (7,10,11).The results obtained in our study indicate that the thymocyte cultivation in vitro conditions leads to apop-tosis of more than a half of the cultivated thymocytes (approximately 58%). Such a high degree of apoptosis is probably the consequence of the thymocyte isola-tion. Actually, a sudden isolation of the nonstimulated thymocytes leads to an abrupt halt of the effect of the thymic microenvironment on thymocytes, as well as to the halt of the effect of many cytokines within thymus itself. All the aforementioned factors can significantly increase a spontaneous apoptosis in the thymocyte cul-ture. Similar results were obtained in Perandones and the associates’ research (12).Our further research showed that high ascorbic acid doses significantly reduced the apoptosis inten-sity. The greatest apoptosis inhibition was detected in the cultures with 10000 µg/ml (p < 0.001). The lowest apoptosis inhibition was detected in the cultures with 100 µg/ml (p < 0.05). A significant inhibition was not detected in the cultures with 10 µg/ml. According to the obtained results, we can conclude that high concen-trations of ascorbic acid significantly inhibit a sponta-neous thymocyte apoptosis. The spontaneous apoptosis inhibition is directly proportional to the ascorbic acid concentration. Campbell and the associates’ earlier re-searches (7) also indicate the protective effect of the ascorbic acid on the spontaneous splenocytes’ apopto-sis. Similar results were issued out by Vissers et al. as-Graph 1. Number of apoptotic Ly cultivated with ascorbic acid for 24hGraph 2. The effect of ascorbic acid on spontaneous  thymocyte apoptosisActa Medica Medianae 2005. Vol. 4  Ascorbic acid modulates spontaneous thymocyte apoptosis23sociates (13). Their results indicate antiapoptotic effect of the ascorbic acid on the activated as well as  nonac-tivated human neutrophils. One of the reasons of such a phenomenon may be that many different ways of apoptosis induction make one common way in which the ascorbic acid manifests its antiapoptotic effect (7). Total reduction of the apoptotic cell number, resulting in the number increase of the T-cell pool, can be one of the mechanisms through which the ascorbic acid has the stimulatory effect on the immune system (13). conclusionApoptosis  represents  a  strictly  controlled  pro-cess. Disturbances in its intensity lead to serious di-sorders. Ascorbic acid has a very significant effect in the intensity modulation of the spontaneous thymocyte apoptosis. Such apoptosis inhibition of the T-cells, as effectory cells of the immune system, can represent one of the basic factors through which the ascorbic acid has stimulatory effect on the immune system.1.  Cirić  O,  Budec  M,  Leposavić  G.  Neuro-endokrino-imunologija.  Beograd:  Zavod  za  udžbenike  i  nastavna sredstva; 2000. 2.  Hale HP, Clark AG, Li J, Greer PK, Kraus VB. Age- related thymic atrophy in the guinea pig. Dev Compa Immunology 2001; 25: 509-18. 3.  Rathmell JC, Thompson CB. Pathways of apoptosis in lymphocyte development, homeostasis and disease. Cell 2002; 109: 97-107. 4.  Penn ND, Purkins L, Kelleher J, Heatley RV. The effect of  dietary  supplementation  with  vitamins A,  C  and  E on cell-mediated immune function in elderly long-stay patients: a randomized controlled trial. Age Ageing 1991; 20: 169-74. 5.  Heuser G, Vojdani A. Enhancement of natural killer cell activity and T and B cell function by buffered vitamin C in patients exposed to toxic chemicals: the role of protein kinase-C.  Immunopharmacol  Immunotoxicol  1997; 19:291-312.6.  Goodwin JS, Brodwick M. Diet, aging, and cancer. Clin Geriat Med 1995; 11: 577-89.7.  Campbell  JD,  Cole  M,  Bunditrutavorn  B,  Vella A.  T. Ascorbic acid is a potent inhibitor of various forms of T cell apoptoris. Cell Immunol 1999; 194: 1-5.8.  Kelley  DS,  Bendich  A.  Essential  nutrients  and immunologic functions. Am J Clin Nutr 1996; 63: 994-6.9.  Bergman M, Salman H, Djaldetti M. In vitro immune response oh human peripheral blood cells to vitamins C and E. J Nutr Biochem 2004; 1: 45-50.10. Hemila H. Vitamin C supplementation and the common cold-was Linus Pauling right or wrong? Int J Vitam Nutr Res 1997; 67:329-35.11. Hemila  H.  Vitamin  C,  respiratory  infections  and  the immune system. Trends Immunol 2003; 24: 579-80.12. Perandones  CE,  Illera VA,  Peckham  D.  Regulation  of apoptosis  in  vitro  in  mature  murine  spleen  T  cells.  J Immunol 1993; 151: 3521-9.13. Vissers MCM, Hampton MB. The role of oxidants and vitamin C on neutrophil apoptosis and clearance. Biochem Soc Transactions 2004; 32: 499-501. ReferencesASKORBINSKA KISELINA MODULIŠE SPONTANU APOPTOZU TIMOcITAVoja Pavlović, Snežana Cekić, Vladmila Bojanić, Nenad Stojiljković i Goran RankovićTokom procesa maturacije, svi timociti podležu procesu pozitivne ili negativne selekcije. Negativna selekcija timocita dovodi do stvaranja tolerancije na sopstvene antigene. Danas se smatra da jedan od načina delovanja askorbinske kiseline jeste modulacija preživljavanja T ćelija, tj. modulacija intenziteta apoptoze. U radu je ispitivan efekat različitih koncentracija askorbinske kiseline na intenzitet spontane apoptoze timocita.Rezultati pokazuju da oko 58% izolovanih timocita podleže spontanoj apoptozi, nakon 24 sata kultivacije u kompletnom medijumu. Kultivacija izolovanih timocita sa različitim koncentracijama vitamina C, pokazuje da se broj apoptotičnih timocita smanjuje, srazmerno povećanju koncentracije askorbinske kiseline. Najdramatičniji efekat je detektovan u kultu-rama sa najvećom koncentracijom vitamina C (10000 µg/ml), gde se procenat apoptotičnih timocita smanjuje na oko 32%. Upoređivanje dobijenih podataka sa vrednostima spontane apoptoze timocita, pokazuje da je statistički najznačajnija inhibicija apoptoze verifikovana u kulturama sa 10000 µg/ml vitamina C.Visoke koncentracije askorbinske kiseline značajno inhibiraju proces spontane apopto-ze timocita. Ovakva inhibicija apoptoze T ćelija, kao efektornih ćelija imunološkog sistema, može predstavljati jedan od osnovnih faktora preko kojih askorbinska kiselina stimulativno utiče na imuni sistem. Acta Medica Medianae 2005;44(4): 21 – 23.Ključne reči: askorbinska kiselina, timus, apoptoza

ASCORBIC ACID MODULATES SPONTANEOUS THYMOCYTE APOPTOSIS

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