Download The effect of the sympathetic nervous system on the production of

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.
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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.
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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.
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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
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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
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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
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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
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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.
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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.
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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.
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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.
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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.
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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
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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
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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
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