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The effect of the sympathetic nervous system on the production of mediators regulating inflammatory processes Thesis of Ph.D. dissertation Zsolt Selmeczy Supervisor: Prof. Dr. E. Szilveszter Vizi Hungarian Academy of Sciences Institute of Experimental Medicine Laboratory of Neuroimmunology and School of Ph.D. Studies Semmelweis University 2005 Budapest INTRODUCTION Both the nervous system and the immune system have an important role in the adaptation of organisms to external and internal environmental challenges. The sympathetic nervous system, in the research of which E. Szilveszter Vizi and his colleagues had a major role, acts as an interface between these two systems. Evidence for the connection of these two systems is the noradrenergic innervation of primary and secondary lymphoid organs, release of neurotransmitters in immune organs, and adrenoceptors expressing on the surface of immune cells, among which β2adrenoceptors (β2ARs) can be found on all types of immune cells except T helper 2 lymphocytes, while the presence of α2-adrenoceptors (α2ARs) is controversial. The immunmodulant effect of the sympathetic nervous system is manifested in the blocking of cellular immunity and the production of proinflammatory cytokines such as tumour necrosis factoralpha (TNF-α), and in the stimulation of humoral immunity and the production of anti-inflammatory cytokines such as interleukin (IL)-10. The functionality of this regulatory system is notably affected by the changeableness of components of two subsystems - the sympathetic nervous system and the immune system. The effect of noradrenaline (NA) transmitting information of the sympathetic nervous system is determined by its amount released from sympathetic varicosities and the duration of its presence in the environment of the target, while immune cells, targets of sympathetic regulation, go through number of differentiating and maturating processes during their life span, which may change their susceptibility to various regulating effects. 2 AIM Since the presence of noradrenalin in extracellular space is regulated by complex mechanisms, and immune cells in an organism go through differentiating and maturating processes during their life span, the aim of this work was to study: • How do the events influencing the extracellular concentration of NA effect the production of mediators regulating inflammatory processes in vivo? • How do the inducibility of inflammatory mediators, and its sympathetic modulation change during differentiation of the immune cells in vitro? METHODS Animals: Wild type mice and their genetically modified NA-transporterdeficient (NET-KO) counterparts were obtained from M. G. Caron’s laboratory. All procedures were carried out in accordance with the European Community’s guidelines concerning the use of experimental animals and those of the institutional ethics committee. Animal experiments: Animals were injected intraperitoneally with drugs at 1 ml/100 g body weight, 60 min before the administration of lipopolysaccharide (LPS, 10 mg/kg, i.p.). Animals were decapitated 90 min after LPS administration and plasma was separated from blood and stored at – 70°C until further analysis. 3 Cell lines and culture conditions: In these experiments we used PLB-985 human myelomonoblastic leukaemia cell line obtained from M. C. Dinauer, which cells were differentiated with 0.5% (v/v) dimethylformamide towards granulocyte, or with 50 nM 1,25-dihydroxyvitamin D3 towards monocyte/macrophage direction for 4 days. After differentiation, cells were plated into 96-well plates and after a 2-hour resting time they were treated with drugs. 24 hours after the treatment inducing cytokine production, plates were centrifuged and supernatants were collected and stored at – 20 °C until analysed. Measurement of cytokines: TNF-α and IL-10 content of plasma and supernatants was determined by ELISA method. Western blot: PLB-985 cells were differentiated as described in “Cell lines and culture conditions”, then were lysed after treatments with drugs. Protein content of lysates was determined by modified Lowry-method. 75 µg protein samples were separated by SDS-PAGE and transferred to PVDF membrane. Membranes were incubated with anti-pERK, anti-pCREB and anti-ERK antibodies. We used the ECL system for chemiluminescence detection. FACS analysis: The differentiation stage of PLB-985 cells was assessed by FACS analysis. CD14, CD16 and CD35 surface markers were detected by staining with phycoerytrin-coupled anti-CD14, fluorescein isothiocyanate (FITC)-coupled anti-CD16, FITC-coupled anti-CD35 or with appropriate isotype controls. Collaborators of Department of Immunology at ELTE and that of Institute of Haematology and Immunology at OGYK helped in implementation of analysis. 4 Statistical analysis: Statistical analysis of data from animal experiments was performed by one-way analysis of variance followed by Dunnett’s test or by a two-tailed Student’s t-test where appropriate P < 0.05 was considered as statistically significant. Statistical analysis of data from in vitro experiments was performed by one-way ANOVA followed by the Tukey-Kramer Multiple Comparison Test where differences were assumed to be significant at P < 0.05. RESULTS Effect of the change of extracellular concentration of NA on the production of inflammatory mediators: • TNF-α and IL-10 production of NA-transporter deficient (NET-KO) animals induced by bacterial endotoxin LPS proved to be lower than those of wild type ones, which denotes the decreased immunological activity of NET-KO animals. • Since activation of α2-adrenoceptors (α2AR) located on noradrenergic axon terminals provokes the inhibition of NA release (negative feedback), we studied the effect of LPS on TNF-α and IL-10 production in the presence of selective α2AR antagonist CH-38083 (7,8methyilenedioxy-14α-hydroxy-alloberbane.HCl). CH-38083 given at doses of 10 mg/kg 60 min prior to LPS treatment decreased LPS-evoked TNF-α and increased IL-10 production in both wild type and NET-KO 5 animals, which indicates the functional integrity of α2ARs in NET-KO mice. • The study of the function of βARs mediating the immunmodulant effect of the sympathetic nervous system showed that the pretreatment with βAR antagonist propranolol (10 mg/kg) significantly increased LPSinduced TNF-α production in both groups, but its extent was more pronounced in the case of NET-KO animals. However, propranolol provided to be inhibitory in case of IL-10 production in both groups of animals, but in this case the effect was more pronounced in wild type animals. • The tricyclic antidepressant desipramine, which blocks NA uptake to axon terminals, given at doses of 10 mg/kg 60 min prior to LPS treatment provided to be effective not only in wild type, but in NET-KO animals too, because it decreased LPS-induced TNF-α, and increased IL-10 production in both groups of mice. Whereas, desipramine is not a selective blocker of NET, this finding suggests that other monoamin transporters may take over partially the function of NET. Results concerning the change of the inducibility of the production of inflammatory mediators and its sympathetic modulation during differentiation of immune cells: • To model the differentiation of immune cells, we used PLB-985 human myelomonoblastic leukaemia cell line, which cells can be differentiated towards granulocyte and monocyte/macrophage direction. To induce TNF-α production of PLB-985 cells, the bacterial chemotactic peptide FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine), opsonized 6 zymosan (OZ) and protein kinase C activator PMA (phorbol 12myristate 13-acetate) were used beside LPS used in in vivo experiments. From among the four inductors, only PMA (100 nM) evoked TNF-α production in immatured PLB-985 cells, while other agents proved to be ineffective. • PLB-985 cells differentiated towards granulocyte direction reacted to PMA non-significantly, while OZ (100 µg/ml) significantly induced TNF-α production. • In case of PLB-985 cells differentiated towards monocyte/macrophage direction, FMLP (100 nM) and LPS (10 µg/ml) provided to be an effective inductor of TNF-α production. • From among the cell surface receptors instrumental in the recognition of inductors, CD14, recognising LPS, was expressed only on cells differentiated towards monocyte/macrophage direction, while CD16, recognising OZ, could be found only on cells differentiated towards granulocyte direction. CD35, recognising FMLP, was expressed on both differentiated cells. • It was also proven that the expression of a surface receptor is not enough in all cases to evoke cell response for an inductor (for example in case of CD35 and FMLP). • We tried to follow the pattern of IL-10, one of the most familiar antiinflammatory cytokine, production during differentiation in vitro, but none of agents used in these experiments could induce significant IL-10 production in either forms of PLB-985 cells. • To model the immunmodulant effect of the sympathetic nervous system experienced in vivo, we used βAR agonist isoproterenol in vitro, which had proved earlier to have immunmodulant effect both in vivo and in 7 vitro. In vitro experiments, its effect was studied by two stimuli. One was LPS used earlier in vivo. Isoproterenol (100 µM) blocked LPSinduced TNF-α production only in cells differentiated towards monocyte/macrophage direction, while in case of the other two celltypes, immunmodulant effect of isoproterenol could not be shown. • The other stimulus was the aspecific immunological inductor PMA. Its choice was justified by that we tried to follow the change of immunmodulation during differentiation of PLB-985 cells, and from among three inductors beside LPS, only PMA could evoke TNF-α production in all three differentiated forms of PLB-985 cells, even if it was not significant in all cases. In contrast to the case of LPS, isoproterenol modulated the effect of PMA in each differentiated stage, but in a different manner: it blocked TNF-α production in nondifferentiated cells, while it increased that in differentiated cells. • Of intracellular proteins having role in the regulation of TNF-α production, amount of pCREB (phosphorilated form of cAMPresponsive element binding protein), which also has role in transmitting signals from βARs, and amount of pERK (phosphorilated form of extracellular signal regulated kinase) mediating the effect of PMA were analysed by Western blot during the differentiation and activation of PLB-985 cells. As there are not data about these proteins in case of PLB-985 cells, our results provide further information about this cell line beside the main aim of this study. From among two proteins, pCREB was expressed in high amount under normal condition, independently from differentiation stage. Only PMA could change it significantly, namely it decreased the level of pCREB in differentiated 8 cells, which was partly inhibited by pretreatment with isoproterenol in both cases. • Under normal conditions, the expression of pERK could not be shown in either non-differentiated or differentiated PLB-985 cells similarly to isoproterenol-treated cells. PMA increased the level of pERK in both non-differentiated and differentiated PLB-985 cells. Isoproterenol decreased this PMA-induced increment of pERK level in nondifferentiated cells, while it increased that in cells differentiated towards granulocyte and monocyte/macrophage direction. 9 CONCLUSIONS Conclusions from the investigation of the effect of changing extracellular concentration of NA on the production of inflammatory mediators: • The biophase concentration of NA, the neurotransmitter of the sympathetic nervous system, and the duration of its presence in biophase space have an important role in the immune response of organisms: it blocks proinflammatory cytokine, TNF-α production, and increases anti-inflammatory cytokine IL-10 production through βAR expressed on immune cells. • This statement is supported and the importance of monoamin transport systems is provided by finding that LPS-evoked production of proinflammatory cytokine TNF-α is decreased in NET-KO animals by the increased biophase level of NA. • We provided that βARs expressed on the surface of immune cells have a crucial role in the inhibition of proinflammatory TNF-α production in case of both wild type and NET-KO mice. • Based on our observations we conclude that acute or chronic hyper- or hypofunction of the sympathetic nervous system may effect the balance of proinflammatory and anti-inflammatory processes with that it changes the susceptibility of the organism to autoimmune, allergic, infectious, or malignant diseases. 10 Conclusions from the investigation of the effect of cell differentiation on the inducibility of inflammatory mediators production and its sympathetic modulation: • The reactivity of immune cells to different immunological inductors changes during differentiation of immune cells, at least in point of TNFα production. • Necessary but not sufficient assumption of change of reactivity to different immunological inductors is changing of the expression pattern of receptors having a crucial role in the recognition of inductors. • Beside the reactivity of immune cells, the sensitivity of cells for modulating effects of the sympathetic nervous system could also change during differentiation, moreover the direction of modulation could change. • Our observation related to the change of immune cells reactivity to immunologically active agents, and their sensitivity for the modulating effect of the sympathetic nervous system during the differentiation of cells could have practical importance in immunological diseases in which the differentiation stage of cells changed, for example in different types of leukaemia, which are classical in this regard. 11 ACKNOWLEDGMENTS I am grateful to Dr. Judit Szelényi for her advice and help. I am thankful to Prof. Dr. E. Szilveszter Vizi, the president of the Hungarian Academy of Sciences, for the possibility to work at the Department of Pharmacology. I also thank Ms. Julianna Benkő, Ms. Katalin Gazsi, Ms. Anita Bagó, and Ms. Katalin Lévai for their excellent technical help, and all my colleagues in our Institute for the good working conditions. I specially thank Dr. Mária Magócsi, Dr. Ágota Apáti, Ms. Anna Brózik and Mr. Zoltán Ondrejó for their help in Western blot analysis, and Dr. Katalin Német, Dr. György Váradi, Dr. Gabriella Sármay and Mr. Dávid Medgyesi for their help in FACS analysis. I thank Gedeon Richter Ltd., Budapest, Hungary, and the Sepsis Budapest Foundation, Hungary for their financial support during my PhD studies. I am grateful to my Parents and my Family for their devotion and incentive with that they gave me power and safe background to concentrate only on my work. 12 OWN PUBLICATIONS Publications in connection with the theme of dissertation Szelényi J., Kiss P.J., Puskás É., Selmeczy Zs., Szelényi M. and Vizi E.S. (2000) Opposite role of α2- and β-adrenoceptors in the modulation of interleukin-10 production in endotoxaemic mice. Neuroreport 11 3565-68. Szelenyi J, Selmeczy Zs. (2002) Immunomodulatory antidepressants. Curr Opin Pharmacol. 2 428-32. effect of Selmeczy Zs, Szelenyi J, Vizi E.S. (2003) Intact noradrenaline transporter is needed for the sympathetic fine-tuning of cytokine balance. Eur J Pharmacol. 469 175-81. Selmeczy Zs., Szelényi J., Német K., and Vizi E.S. (2003) The inducibility of TNF-α production is different in the granulocytic and monocytic differentiated forms of wild type and CGD-mutant PLB-985 cells. Immunol and Cell Biol. 81 472-9. Publications in other theme Vizi E.S., Szelenyi J., Selmeczy Zs., Papp Z., Nemeth Z.H., Hasko G. (2001) Enhanced tumor necrosis factor-alpha-specific and decreased interleukin-10-specific immune responses to LPS during the third trimester of pregnancy in mice. J Endocrinol. 171 355-61. Rigó J., Szelényi J., Selmeczy Zs., Papp Z., and Vizi E.S. (2004) Spontaneous and endotoxin-induced TNF-α production change inversely in pregnancy. Eur. J. Obstet.&Gynecol. and Reprod. Biol. 114 236-8. 13 More important lectures and posters Selmeczy Zs., Szelényi J., and Vizi E.S. Study of the innate defense system on immature and mature macrophages of mice. JOINT MEETING OF THE PHYSIOLOGICAL SOCIETY AND THE HUNGARIAN PHYSIOLOGICAL SOCIETY, Budapest, 27-29th May 2000, poster Szelényi J., Selmeczy Zs. and Vizi E.S. Changes in the innate immune system due to macrophage maturation EFIS 2000 EUROPEAN FEDERATION OF IMMUNOLOGICAL SOCIETIES, Poznan, 23-27th September 2000, poster Selmeczy Zs., Szelényi J., Vizi E. Sz. A veleszületett immunvédekezés vizsgálata éretlen és érett egér makrofág sejteken. JUBILEE (XXX.) MEETING OF THE HUNGARIAN SOCIETY FOR IMMUNOLOGY, Budapest, 25-27th October 2000, lecture Selmeczy Zs., Szelényi J., Kiss J. P., Haskó Gy., Németh Z., Vizi E. Sz. Az α2-adrenoceptorok szabályozó szerepe az interleukin-10 termelésben endotoxémiás egerekben A MAGYAR IDEGTUDOMÁNYI TÁRSASÁG VIII. KONFERENCIÁJA, Szeged, 24-27th January 2001, poster Selmeczy Zs., Szelényi J. and Vizi E.S. Different sensitivity of mature and immature macrophages for immunomodulation. 3RD MEETING OF THE FEDERATION OF THE EUROPEAN PHARMACOLOGICAL SOCIETIES, Lyon, 6-9th July 2001, poster Szelenyi J., Selmeczy Zs. and Vizi E.S. Moxonidine shifts the Th1/Th2 cytokine balance in endotoxaemic mice 3RD MEETING OF THE FEDERATION OF THE EUROPEAN PHARMACOLOGICAL SOCIETIES, Lyon, 6-9th July 2001, poster Szelényi J., Selmeczy Zs. and Vizi E.S. Immunomodulatory effect of moxonidine, a new antihypertensive agent 6TH INT. CONGR. IN NEUROIMMUNOLOGY, Edinburgh, 3-7th September 2001, poster 14 Selmeczy Zs., Szelényi J., Németh K., Dinauer M.C., Vizi E.Sz. A TNF-α indukció jelentősége a szuperoxid-termelés hiányával járó immunológiai rendellenességben (CGD) A MAGYAR IMMUNOLÓGIAI TÁRSASÁG XXXI. KONFERENCIÁJA, Eger, 17-19th October 2001, poster Selmeczy Zs., Szelényi J., Német K. and Vizi E.S. Altered cytokine response in the wild-type and the chronic granulomatous disease- (CGD)-mutant PLB-985 cells XIVTH WORLD CONGRESS OF PHARMACOLOGY, San Francisco, 712th July 2002, poster Szelényi J., Selmeczy Zs., and Vizi E.S. Impaired NA reuptake affects cytokine production XIVTH WORLD CONGRESS OF PHARMACOLOGY, San Francisco, 712th July 2002, poster Szelényi J., Selmeczy Zs., and Vizi E.S. Study of the interaction between the monoamine- and inflammatorysystems in depression 5TH ISNIM CONGRESS, Montpellier, 9-11th September 2002, poster Selmeczy Zs., Szelényi J., Magócsi M., Apáti Á. és Vizi E.Sz. Érett és éretlen makrofágok eltérő érzékenysége az immunomodulációra A MAGYAR IMMUNOLÓGIAI TÁRSASÁG XXXII. KONGRESSZUSA, Kaposvár, 30th September- 2nd October 2002, poster Szelényi J., Selmeczy Zs., Vizi E.Sz. A desipramin immunmoduláns szerepének vizsgálata WT-129 és NET-KO egereken A MAGYAR KÍSÉRLETI ÉS KLINIKAI FARMAKOLÓGIAI TÁRSASÁG KONGRESSZUSA, Debrecen, 12-14th December 2002, poster Selmeczy Zs., Szelényi J., Magócsi M., Apáti Á. és Vizi E.Sz. Érett és éretlen makrofágok eltérő érzékenysége a szimpatikus idegrendszer immunomoduláns hatására A MAGYAR IDEGTUDOMÁNYI TÁRSASÁG IX. KONFERENCIÁJA, Balatonfüred, 23-25th January 2003, poster 15 Szelényi J., Rigó J., Papp Z., Selmeczy Zs. and Vizi E.S. Dichotomy of TNF-α production in pregnancy EFIS 2003, Rhodosz, 8-12th June 2003, poster Selmeczy Zs., Szelényi J., Vizi E.S. The role of norepinephrine-reuptake in lipopolysaccharide-evoked tumor necrosis factor-alpha and interleukin-10 response A MAGYAR IMMUNOLÓGIAI TÁRSASÁG XXXIII. KONGRESSZUSA, Győr, 15-17th October 2003, lecture and poster Selmeczy Zs., Szelényi J., Rigó J., Bálint K.B., Papp Z., Vizi E.Sz. A spontán és az indukált citokintermelés változásai terhesség során IMMUNFARMAKOLÓGIAI KONFERENCIA, Debrecen, 11-13th December 2003, lecture Selmeczy Zs., Szelényi J., Rigó J., Bálint K.B., Papp Z., Vizi E.Sz. Változások A spontán és az indukált citokintermelésben és annak szimpatikus szabályozásában terhesség során XXXIV. MEMBRÁN-TRANSZPORT KONFERENCIA, Sümeg, 1-4th June 2004, poster Szelényi J., Selmeczy Zs., Vizi E.S. Effect of monoamine transporter inhibitors on the LPS induced cytokine production EPHAR 4TH MEETING, Porto, 14-17th July 2004, lecture and poster 16