BACKGROUND
almost two decades ago the observation has been made that lymphoid organs are directly innervated, mostly by neuropeptidergic fibers, and the question was raised whether the supplied neurotransmitters might have immunomodulatory functions  <cit> .
this finding provided an anatomical rationale for the investigation of the effects of neurotransmitters on leukocytes, especially on b and t lymphocytes.
it turned out, that several neurotransmitters have very distinct and varying functions on different leukocyte subsets .
however, up to now there is no clear pattern of how the neuro-endocrine system in its function as the superordinate regulatory organ of the body modulates the immune system in common.
this is due to the complexity of both organ systems and their multilayer interaction.
consequently, the discussion is still ongoing if and how emotions and sensations are translated into a general stimulation or suppression of the immune system.
nevertheless, a large number of reports have been published that describe the function of neurotransmitters on certain leukocytes.
best characterized is probably the function of norepinephrine.
this neurotransmitter is of special interest, since it is not only locally released from sympathetic nerve cells, but is also systemically disseminated after release from the adrenal gland.
furthermore, catecholaminergic innervation of lymph nodes increases under psycho-social stress conditions, as was shown on macaques  <cit> .
t and b lymphocytes both express the β2-adrenoceptor, which is responsible for the intracellular signal transduction of norepinephrine.
however, it is unclear whether both activated t helper  <dig> and th <dig> lymphocytes, or only activated th <dig> lymphocytes express the β2-adrenoceptor  <cit> .
in th <dig> lymphocytes, norepinephrine has influence on the expression of interferon -γ, depending on the time-point of its presence during activation: when norepinephrine was added before activation, ifn-γ production decreased; when added after activation, ifn-γ production increased  <cit> .
the importance of this neuro-immunologic axis becomes even more obvious in patients with spinal cord injury, which have an impaired response to infections.
in a mouse model it has been shown that, depending on the level of spinal cord injury, increased concentrations of circulating corticosterone and norepinephrine are present, which lead to an impaired antibody synthesis  <cit> .
however, β2-adrenergic stimulation or cyclic adenosine-monophosphate  accumulation - which is a key signalling event caused by this receptor - elicit in concert with other stimuli divergent effects in b cell subsets concerning proliferation, b7- <dig> and major histocompatibility complex ii expression, differentiation to antibody-secreting cells, and antibody production  <cit> .
interestingly, the antibody production largely depends on the duration of camp accumulation.
short term elevation of the cellular camp concentration results in an increase of antibody production, whereas a long term elevation decreases antibody production  <cit> .
a recent work by grebe et al.
reported that β-blockers such as nadolol enhance antiviral cd8+ t lymphocyte responses in mice, suggesting an immuno-suppressive effect of norepinephrine  <cit> .
dopamine is the metabolic precursor of norepinephrine.
the secondary lymphoid tissues are abundantly innervated by the sympathetic nerves that store a large amount of dopamine  <cit> .
the d <dig> to d <dig> receptors for dopamine are differentially expressed on leukocyte subsets  <cit> , and it has also been reported that dopamine can act via β-adrenoceptors  <cit> .
dopamine suppresses the proliferation and cytotoxicity of t lymphocytes  <cit> , but stimulates adhesion to fibronectin  <cit> .
with regard to the cytokine release, the function of dopamine is not clear.
depending on the experimental setting, dopamine has been reported to have increasing  <cit>  as well as decreasing  <cit>  function.
in the first study, the peripheral blood mononuclear cell  fraction was activated with anti-cd <dig> and anti-cd <dig> antibodies  <cit> .
in the second study, the t lymphocytes were further purified from the pbmc fraction and activated only with an anti-cd <dig> antibody  <cit> .
substance p is a peptide of the neurokinin family, which plays a role in depressive disorders  <cit> , and inflammatory processes  <cit> .
substance p is also released by peripheral nerve endings that innervate lymphoid organs  <cit> .
in contrast to dopamine, substance p inhibits t lymphocyte adhesion to fibronectin  <cit> , and in contrast to norepinephrine, substance p increases the number of immunglobulin-secreting cells  <cit> .
unfortunately, there exists no general scheme to predict how neurotransmitter will act on leukocytes, in contrast the effect of a neurotransmitter depends not only on the parameter of interest but also on the cell type investigated and its activation state or phenotype as well as on the lymphoid tissue from which the cells derive  <cit> .
this brief introduction shows that norepinephrine, dopamine and substance p have diverse, sometimes opposite effects on b and th lymphocytes.
in comparison to these lymphocyte subpopulations, the influence of neurotransmitters on the function of cd8+ t lymphocytes is much less investigated.
therefore, we conducted the present study in order to complement the picture of neurotransmitter action on leukocyte subsets.
the cd8+ t cells have to be activated in the lymph node by their t cell receptor.
they then emigrate from the lymph node, extravasate from the blood stream at infected or inflamed tissue sites and migrate through the tissue in search for target cells.
after recognition of these cells, they eliminate these cells.
we investigated the influence of norepinephrine, dopamine and substance p on these key functions of cd8+ t lymphocytes in vitro: activation, migration, extravasation and cytotoxicity.
beyond this, we provide molecular explanations on how the neurotransmitters affect these cells functions.
RESULTS
impact of neurotransmitters on the activation of cd8+ t lymphocytes depends on il-2
cd8+ t lymphocytes were activated by cross-linking of the t cell receptor molecules cd <dig> and cd <dig> by immobilized antibodies.
activation was monitored by staining of the classical activation markers cd45r <dig> and cd <dig>  after four days of activation  <dig>  ±  <dig> % of the cd8+ t lymphocytes were double positive for cd45r <dig> and cd <dig> .
norepinephrine and dopamine inhibited the activation to  <dig>  ±  <dig> % and  <dig>  ±  <dig> % of the cells, respectively, whereas both effects were significant .
substance p led only to a slight, non-significant reduction .
this inhibition of activation was not due to cell death, as we have analyzed by flow-cytometry.
il- <dig> is a well known cytokine in t cell activation, and consequently the exogenous addition of il- <dig> strongly supports the activation process.
the stepwise addition of il- <dig>  had two effects: firstly, the more il- <dig> was added, the weaker the inhibitory effects of the neurotransmitters were.
secondly, the number of cd45r <dig> and cd <dig> double-positive t lymphocytes increased dose-dependently .
none of the investigated neurotransmitters norepinephrine, dopamine or substance p had a significant influence on the activation in the presence of  <dig> ng/ml il- <dig> .
thus, we asked, whether the neurotransmitters might have an effect on the il- <dig> expression.
we analyzed the amount of il- <dig> mrna by semi-quantitative rt-pcr after  <dig> hours of activation .
cells treated with norepinephrine  and dopamine  showed a decrease in their level of il- <dig> mrna to 31% and 27%, respectively, when compared to control cells and adjusted to β-actin expression.
substance p treatment  led to a moderate reduction to 86%, whereas non-activated cells showed only 18% expression of il- <dig> rna when compared to their activated counterparts .
the rt-pcr results were further verified by il- <dig> elisa experiments to investigate if protein expression and secretion are also decreased.
therefore the cell culture supernatant after two days of activation was collected.
under control conditions cd8+ t lymphocytes secreted  <dig>  ±  <dig>  pg il- <dig> per  <dig> ×  <dig> cells per day .
in the presence of neurotransmitters the amount of secreted il- <dig> was decreased .
unactivated lymphocytes secreted only a small amount of il- <dig>
these results deliver an explanation for the reduction of activation by the neurotransmitters, and it explains, why no effects of the neurotransmitters were observed, when il- <dig> was added exogenously during activation.
norepinephrine and dopamine inhibit the il- <dig> expression thus interfering with the generation of an autocrine il- <dig> loop necessary for optimal activation of t cells.
the il- <dig> promoter is under the control of several transcription factors.
we thus investigated, whether the neurotransmitters might inhibit one or more of these transcription factors.
activator protein - <dig>  nuclear factor of activated t-cells , nuclear factor -κb, and the camp response element binding protein  are known to be necessary for optimal il- <dig> gene transcription  <cit> .
phosphorylated erk1/ <dig> leads to an up-regulation of c-fos expression  <cit>  and c-fos in turn, when dimerized with the jun family of transcription factors, forms ap- <dig>  <cit> .
therefore, we investigated erk1/ <dig> phosphorylation as a marker for ap- <dig> activity .
erk1/ <dig> phosphorylation was strongly reduced by norepinephrine  and dopamine  and to a minor degree by substance p .
in the non-activated control, no erk1/ <dig> phosphorylation was detectable.
we next investigated the phosphorylation of the transcription factors p <dig>  and creb one hour after activation.
both of these transcription factors were highly phosphorylated in activated cells, and no differences in the induction of phosphorylation were observed with any of the used neurotransmitters .
nf-κb is under the control of iκb, which restricts nf-κb to the cytoplasm and inhibits its dna binding activity  <cit> .
upon activation of the ctl, iκb is phosphorylated and subsequently degraded , thereby releasing nf-κb and allowing its transition to the nucleus .
when adjusted to the whole amount of iκb, norepinephrine reduced the phosphorylation to 42%, dopamine to 46%, and substance p to 37% .
due to this, a reduced degradation was detected in activated cd8+ t lymphocytes.
compared to non-activated cells, only few iκb  was detected in activated cells.
under neurotransmitter treatment, the remaining amount of iκb increased to 49% , 36% , and 35% .
in consequence, a reduced amount of phosphorylated nf-κb was found in the nuclear fraction of the lymphocytes when treated with the neurotransmitters during activation: 39% with norepinephrine, 58% with dopamine, and 55% with substance p .
transcription factors of the nfat family are activated by calcium signalling via calmodulin/calcineurin.
an involvement of nfat in the inhibition of il- <dig> expression is unlikely, since none of the neurotransmitters had a significant influence on the intracellular calcium increase in response to cd3/cd <dig> cross-linking .
t cell activation via cd <dig> and cd <dig> leads to activation of associated kinases of the src-family and the recruitment and activation of the tyrosine kinase zap- <dig> .
with regard to il- <dig> transcription two key signalling pathways become activated.
phospholipase c gamma  is recruited to the membrane site and becomes activated.
plcγ cleaves phosphatidylinositol bisphosphate  to yield inositol triphosphate  and diacylglycerol .
dag activates nfκb via protein kinase θ and ip <dig> opens intracellular calcium storages.
the increased level of intracellular ca2+ activates the serine phosphatase calcineurin, which dephosphorylates nfat.
dephosphorylated nfat enters the nucleus.
cd <dig> and cd <dig> signalling leads to the activation of ras.
ras induces the activation of the transcription factor ap- <dig> via the map kinase cascade raf-1/mek/erk and further mediates the degradation of iκb via akt.
in summary we could show that those transcription factors, which are under the control of ras, i.e.
ap- <dig> and iκb, are regulated by neurotransmitters  <cit> , whereas those, which are regulated by downstream signals of the plcγ, i.e.
nf-κb and nfat, are not affected  <cit> .
effect of neurotransmitters on the migratory behaviour
activated cd8+ t lymphocytes showed a striking and significant  increase of the migratory activity from  <dig>  ±  <dig> % to  <dig>  ±  <dig> % locomoting cells after activation .
furthermore, we have reported previously, that the migratory activity of naïve, non-activated cd8+ t cells is increased by the treatment with norepinephrine or substance p  <cit> .
likewise, dopamine significantly  increased the migratory activity from  <dig>  ±  <dig> % to  <dig>  ±  <dig> % locomoting cells .
in contrast to these results, the migratory activity of activated t lymphocytes was reduced by norepinephrine and substance p, whereas dopamine had no effect .
norepinephrine almost significantly  reduced the migration of activated cd8+ t cells from  <dig>  ±  <dig> % to  <dig>  ±  <dig> % locomoting cells .
dopamine had no effect on the migratory activity .
substance p reduced the migration from  <dig>  ±  <dig>  to  <dig>  ±  <dig> % locomoting cells ; this effect was however not significant due to the high standard deviation.
these differences might be due to the fact, that dopamine receptors are down-regulated on activated cd8+ t lymphocytes as we found by western blot analysis, whereas the β1-adrenoceptor and the nk- <dig> receptor are still expressed by activated cells .
we found only the d <dig> and d <dig> receptors and to a lesser degree the d <dig> receptor to be expressed on naïve cd8+ t lymphocytes.
all three of them become down-regulated during t cell activation and with the exception of the d <dig> receptor these findings are in accordance with watanabe et al.
<cit> .
thus, naïve and activated cd8+ t lymphocytes differ in their level of spontaneous migratory activity.
in the naïve state the spontaneous migratory activity is only moderate whereas in the activated state more than half of the cells show migratory activity.
this observation is not surprising since the expression pattern of matrix adhesion molecules changes during the activation process and thereby the intracellular signalling.
consequently, neurotransmitters have distinct effects on naïve and activated cd8+ t lymphocytes.
collagen i receptors such as very late antigen - <dig> and vla- <dig> are almost absent on naïve cd8+ t cells but become upregulated during activation  <cit> .
this might explain our observation that the naïve lymphocytes show less spontaneous migratory activity than the activated lymphocytes in our migration assay containing 95-98% collagen i as almost single matrix component, with the remainder being comprised of type iii collagen.
naïve cd8+ t lymphocytes show an increase in their migratory activity in response to dopamine , norepinephrine and substance p  <cit> , with dopamine being the strongest inducer.
our finding on dopamine is consistent with watanabe et al.
<cit> , who described that dopamine induces chemotaxis in cd45ra+ naïve cd8+ cells via d <dig> receptor and postulated a role for endogenous dopamine in the homing of naïve cd8+ t cells.
adhesion of ctl to endothelium is selectively regulated by neurotransmitters
the ability to extravasate is an important property of immune cells independent of their activation state.
naïve lymphocytes need to extravasate during the homing process and activated cells extravasate in order to reach tissue sites of inflammation or injury.
the idea that neurotransmitters influence the extravasation process seems likely, because nerve fibres are particularly concentrated around vascular endothelial cells  <cit> .
non-activated cd8+ t lymphocytes show only low adhesion to the endothelium, but a high rolling activity, as we have investigated by our flow-through adhesion assay .
in activated cd8+ t lymphocytes, the adhesion significantly  increased from  <dig>  ±  <dig>  to  <dig>  ±  <dig>  cells .
due to the increased adhesion, the rolling activity is reduced.
both adhesion and rolling of activated cd8+ cells is increased by addition of each of the investigated neurotransmitters directly to the experiment.
norepinephrine significantly  increased the adhesion of the activated cells from  <dig> ±  <dig> to  <dig> ±  <dig> cells .
likewise, the rolling was significantly  increased from  <dig> ±  <dig> to  <dig> ±  <dig> cells/min .
dopamine and substance p enhanced the adhesion and rolling of the activated cells only marginally, which was not statistically significant .
with regard to naïve, non-activated cd8+ cells, all of the investigated neurotransmitters had only minor effects.
however, dopamine significantly  increased the adhesion of the naïve, non-activated cells , whereas substance p led to a significant reduction .
with regard to dopamine, the finding is in accordance with results from other groups who described an increase in the adhesion of naïve cd45ra+ cd8+ lymphocytes to fibronectin and icam- <dig> in response to dopamine but not substance p  <cit> .
this effect was exclusively mediated by the d <dig> receptor and led to an activation of the fibronectin receptors vla- <dig> and vla- <dig>  <cit> , and the intercellular adhesion molecule - <dig> receptor lymphocyte function-associated antigen - <dig>  <cit>  on naïve lymphocytes.
thus dopamine seems to exhibit a unique function leading to the activation of integrin molecules in naïve cd8+ cells which cannot be induced by norepinephrine or substance p. this observation supports the theory of dopamine being a specific homing factor of naïve cd45ra+ cd8+ cells  <cit> .
since the neurotransmitters were added directly to the cell suspension at the beginning of each experiment both cell types involved - the lympocytes as well as the endothelial cells- are exposed to the substance.
interestingly, norepinephrine, but not dopamine or substance p induced a significant  increase of the il- <dig> release from the endothelial cells , even when pre-activated with the cytokine il-1β .
chemokines such as il- <dig> are cationic proteins, which bind to heparansulfate and related glycosaminoglycan moieties on the endothelial monolayer  <cit> , and thereby becomes presented to rolling cells.
several studies have conclusively shown that the recruitment of circulating leukocytes at vascular sites in target tissue is linked to activation of gi-protein signalling in leukocytes induced by chemokines presented on the apical site of the endothelial monolayer .
these immobilized chemokines are involved in the rapid modulation of the avidity of integrins such as vla- <dig>  <cit>  or lfa- <dig> expressed by lymphocytes.
to confirm this hypothesis, we investigated the surface expression of the according il- <dig> receptors cxcr <dig> and cxcr <dig> on cd8+ lymphocytes: cxcr <dig> was almost absent on naïve cells , but it became upregulated during activation .
we did not detect an expression of the il- <dig> receptor cxcr <dig> neither on naïve nor on activated cd8+ t lymphocytes , which corresponds to the results of takata et al.
<cit>  and nishimura et al.
<cit> , but is in conflict with other findings  <cit> .
to investigate a possible role for cxcr <dig> in the adhesion of cd8+ t lymphocytes to endothelium in the presence of norepinephrine we blocked this receptor, and cxcr <dig> as well , via a specific blocking antibody .
this blockade led to a significant  reduction of the activated cd8+ t lymphocyte adhesion to the endothelial cells in the presence of norepinephrine compared to the isotype  control from  <dig> ±  <dig> to  <dig> ±  <dig> adherent cells.
this reduction was however not completely down to the control level .
thus, the effect of norepinephrine on the adhesion of activated cd8+ t lymphocytes shown in fig.
5a is not only mediated by il- <dig>  instead the effect can be further mediated by other chemokines released by the endothelium or by a direct effect of norepinephrine on the t cells.
since we have just screened a subset of chemokines released by endothelial cells, we cannot exclude that the release of further chemokines is affected by norepinephrine.
from our experiments, we can exclude that substance p and dopamine as well act in the way as norepinephrine does, since these neurotransmitters do not result in an increased release of il- <dig> from the endothelium.
degranulation is not influenced by neurotransmitters
the effects of the neurotransmitters on the ability of the cells to degranulate were measured by β hexosamidase release  in response to cd <dig> cross-linking, which mimics the recognition of a target cells without cd <dig> co-signalling.
this process results in a calcium-dependent release of cytotoxic enzymes from intracellular granules that have been formed during activation.
in naïve cd8+ t lymphocytes, the cd3-induced release of β hexosamidase is as high as the release induced by an isotypic control antibody .
after activation, the cd3-induced release of β hexosamidase is significantly  increased from  <dig>  ±  <dig> % to  <dig>  ±  <dig> % release.
however, none of the investigated neurotransmitters caused any effect in activated cd8+ t lymphocytes .
determination of the calcium-signalling in response to cd <dig> cross-linking showed no differences, too .
in conclusion, it seems that the investigated neurotransmitters can affect the development of armed effector cells, but when these cells once have been formed, they cannot be inhibited in their effectory function by neurotransmitters.
CONCLUSIONS
although we have large and detailed knowledge how certain neurotransmitters act on particular leukocyte subpopulations, the remaining question is still how emotions and sensations are translated into modulatory effects of the immune system, and whether this at all plays a role in the function of the immune system  <cit> .
taken together, we have addressed the question how the neurotransmitters norepinephrine, dopamine and substance p influence the function of cd8+ t lymphocytes.
we have identified a regulation pattern  showing that neurotransmitters can act either immunostimulatory or immunosuppressive.
this depends mainly on the activation state of the t cells.
we were able to assign downstream targets of the observed effects.
neurotransmitters cannot per se induce new functions in cd8+ t lymphocytes, i.e.
migration, adhesion and cytotoxicity, but they play an important role in the fine-tuning of t cell responses.

 almost two decades ago the observation has been made that lymphoid organs are directly innervated, mostly by neuropeptidergic fibers, and the question was raised whether the supplied neurotransmitters might have immunomodulatory functions. this finding provided an anatomical rationale for the investigation of the effects of neurotransmitters on leukocytes, especially on b and t lymphocytes. it turned out, that several neurotransmitters have very distinct and varying functions on different leukocyte subsets. however, up to now there is no clear pattern of how the neuro-endocrine system in its function as the superordinate regulatory organ of the body modulates the immune system in common. this is due to the complexity of both organ systems and their multilayer interaction. consequently, the discussion is still ongoing if and how emotions and sensations are translated into a general stimulation or suppression of the immune system. nevertheless, a large number of reports have been published that describe the function of neurotransmitters on certain leukocytes. best characterized is probably the function of norepinephrine. this neurotransmitter is of special interest, since it is not only locally released from sympathetic nerve cells, but is also systemically disseminated after release from the adrenal gland. furthermore, catecholaminergic innervation of lymph nodes increases under psycho-social stress conditions, as was shown on macaques. t and b lymphocytes both express the β2-adrenoceptor, which is responsible for the intracellular signal transduction of norepinephrine. however, it is unclear whether both activated t helper and th lymphocytes, or only activated th lymphocytes express the β2-adrenoceptor. in th lymphocytes, norepinephrine has influence on the expression of interferon -γ, depending on the time-point of its presence during activation: when norepinephrine was added before activation, ifn-γ production decreased; when added after activation, ifn-γ production increased. the importance of this neuro-immunologic axis becomes even more obvious in patients with spinal cord injury, which have an impaired response to infections. in a mouse model it has been shown that, depending on the level of spinal cord injury, increased concentrations of circulating corticosterone and norepinephrine are present, which lead to an impaired antibody synthesis. however, β2-adrenergic stimulation or cyclic adenosine-monophosphate accumulation - which is a key signalling event caused by this receptor - elicit in concert with other stimuli divergent effects in b cell subsets concerning proliferation, b7- and major histocompatibility complex ii expression, differentiation to antibody-secreting cells, and antibody production. interestingly, the antibody production largely depends on the duration of camp accumulation. short term elevation of the cellular camp concentration results in an increase of antibody production, whereas a long term elevation decreases antibody production. a recent work by grebe et al. reported that β-blockers such as nadolol enhance antiviral cd8+ t lymphocyte responses in mice, suggesting an immuno-suppressive effect of norepinephrine. dopamine is the metabolic precursor of norepinephrine. the secondary lymphoid tissues are abundantly innervated by the sympathetic nerves that store a large amount of dopamine. the d to d receptors for dopamine are differentially expressed on leukocyte subsets, and it has also been reported that dopamine can act via β-adrenoceptors. dopamine suppresses the proliferation and cytotoxicity of t lymphocytes, but stimulates adhesion to fibronectin. with regard to the cytokine release, the function of dopamine is not clear. depending on the experimental setting, dopamine has been reported to have increasing as well as decreasing function. in the first study, the peripheral blood mononuclear cell fraction was activated with anti-cd and anti-cd antibodies. in the second study, the t lymphocytes were further purified from the pbmc fraction and activated only with an anti-cd antibody. substance p is a peptide of the neurokinin family, which plays a role in depressive disorders, and inflammatory processes. substance p is also released by peripheral nerve endings that innervate lymphoid organs. in contrast to dopamine, substance p inhibits t lymphocyte adhesion to fibronectin, and in contrast to norepinephrine, substance p increases the number of immunglobulin-secreting cells. unfortunately, there exists no general scheme to predict how neurotransmitter will act on leukocytes, in contrast the effect of a neurotransmitter depends not only on the parameter of interest but also on the cell type investigated and its activation state or phenotype as well as on the lymphoid tissue from which the cells derive. this brief introduction shows that norepinephrine, dopamine and substance p have diverse, sometimes opposite effects on b and th lymphocytes. in comparison to these lymphocyte subpopulations, the influence of neurotransmitters on the function of cd8+ t lymphocytes is much less investigated. therefore, we conducted the present study in order to complement the picture of neurotransmitter action on leukocyte subsets. the cd8+ t cells have to be activated in the lymph node by their t cell receptor. they then emigrate from the lymph node, extravasate from the blood stream at infected or inflamed tissue sites and migrate through the tissue in search for target cells. after recognition of these cells, they eliminate these cells. we investigated the influence of norepinephrine, dopamine and substance p on these key functions of cd8+ t lymphocytes in vitro: activation, migration, extravasation and cytotoxicity. beyond this, we provide molecular explanations on how the neurotransmitters affect these cells functions.  impact of neurotransmitters on the activation of cd8+ t lymphocytes depends on il-2 cd8+ t lymphocytes were activated by cross-linking of the t cell receptor molecules cd and cd by immobilized antibodies. activation was monitored by staining of the classical activation markers cd45r and cd after four days of activation ± % of the cd8+ t lymphocytes were double positive for cd45r and cd. norepinephrine and dopamine inhibited the activation to ± % and ± % of the cells, respectively, whereas both effects were significant. substance p led only to a slight, non-significant reduction. this inhibition of activation was not due to cell death, as we have analyzed by flow-cytometry. il- is a well known cytokine in t cell activation, and consequently the exogenous addition of il- strongly supports the activation process. the stepwise addition of il- had two effects: firstly, the more il- was added, the weaker the inhibitory effects of the neurotransmitters were. secondly, the number of cd45r and cd double-positive t lymphocytes increased dose-dependently. none of the investigated neurotransmitters norepinephrine, dopamine or substance p had a significant influence on the activation in the presence of ng/ml il-. thus, we asked, whether the neurotransmitters might have an effect on the il- expression. we analyzed the amount of il- mrna by semi-quantitative rt-pcr after hours of activation. cells treated with norepinephrine and dopamine showed a decrease in their level of il- mrna to 31% and 27%, respectively, when compared to control cells and adjusted to β-actin expression. substance p treatment led to a moderate reduction to 86%, whereas non-activated cells showed only 18% expression of il- rna when compared to their activated counterparts. the rt-pcr results were further verified by il- elisa experiments to investigate if protein expression and secretion are also decreased. therefore the cell culture supernatant after two days of activation was collected. under control conditions cd8+ t lymphocytes secreted ± pg il- per × cells per day. in the presence of neurotransmitters the amount of secreted il- was decreased. unactivated lymphocytes secreted only a small amount of il- these results deliver an explanation for the reduction of activation by the neurotransmitters, and it explains, why no effects of the neurotransmitters were observed, when il- was added exogenously during activation. norepinephrine and dopamine inhibit the il- expression thus interfering with the generation of an autocrine il- loop necessary for optimal activation of t cells. the il- promoter is under the control of several transcription factors. we thus investigated, whether the neurotransmitters might inhibit one or more of these transcription factors. activator protein - nuclear factor of activated t-cells, nuclear factor -κb, and the camp response element binding protein are known to be necessary for optimal il- gene transcription. phosphorylated erk1/ leads to an up-regulation of c-fos expression and c-fos in turn, when dimerized with the jun family of transcription factors, forms ap-. therefore, we investigated erk1/ phosphorylation as a marker for ap- activity. erk1/ phosphorylation was strongly reduced by norepinephrine and dopamine and to a minor degree by substance p. in the non-activated control, no erk1/ phosphorylation was detectable. we next investigated the phosphorylation of the transcription factors p and creb one hour after activation. both of these transcription factors were highly phosphorylated in activated cells, and no differences in the induction of phosphorylation were observed with any of the used neurotransmitters. nf-κb is under the control of iκb, which restricts nf-κb to the cytoplasm and inhibits its dna binding activity. upon activation of the ctl, iκb is phosphorylated and subsequently degraded, thereby releasing nf-κb and allowing its transition to the nucleus. when adjusted to the whole amount of iκb, norepinephrine reduced the phosphorylation to 42%, dopamine to 46%, and substance p to 37%. due to this, a reduced degradation was detected in activated cd8+ t lymphocytes. compared to non-activated cells, only few iκb was detected in activated cells. under neurotransmitter treatment, the remaining amount of iκb increased to 49%, 36%, and 35%. in consequence, a reduced amount of phosphorylated nf-κb was found in the nuclear fraction of the lymphocytes when treated with the neurotransmitters during activation: 39% with norepinephrine, 58% with dopamine, and 55% with substance p. transcription factors of the nfat family are activated by calcium signalling via calmodulin/calcineurin. an involvement of nfat in the inhibition of il- expression is unlikely, since none of the neurotransmitters had a significant influence on the intracellular calcium increase in response to cd3/cd cross-linking. t cell activation via cd and cd leads to activation of associated kinases of the src-family and the recruitment and activation of the tyrosine kinase zap-. with regard to il- transcription two key signalling pathways become activated. phospholipase c gamma is recruited to the membrane site and becomes activated. plcγ cleaves phosphatidylinositol bisphosphate to yield inositol triphosphate and diacylglycerol. dag activates nfκb via protein kinase θ and ip opens intracellular calcium storages. the increased level of intracellular ca2+ activates the serine phosphatase calcineurin, which dephosphorylates nfat. dephosphorylated nfat enters the nucleus. cd and cd signalling leads to the activation of ras. ras induces the activation of the transcription factor ap- via the map kinase cascade raf-1/mek/erk and further mediates the degradation of iκb via akt. in summary we could show that those transcription factors, which are under the control of ras, i.e. ap- and iκb, are regulated by neurotransmitters, whereas those, which are regulated by downstream signals of the plcγ, i.e. nf-κb and nfat, are not affected. effect of neurotransmitters on the migratory behaviour activated cd8+ t lymphocytes showed a striking and significant increase of the migratory activity from ± % to ± % locomoting cells after activation. furthermore, we have reported previously, that the migratory activity of naïve, non-activated cd8+ t cells is increased by the treatment with norepinephrine or substance p. likewise, dopamine significantly increased the migratory activity from ± % to ± % locomoting cells. in contrast to these results, the migratory activity of activated t lymphocytes was reduced by norepinephrine and substance p, whereas dopamine had no effect. norepinephrine almost significantly reduced the migration of activated cd8+ t cells from ± % to ± % locomoting cells. dopamine had no effect on the migratory activity. substance p reduced the migration from ± to ± % locomoting cells; this effect was however not significant due to the high standard deviation. these differences might be due to the fact, that dopamine receptors are down-regulated on activated cd8+ t lymphocytes as we found by western blot analysis, whereas the β1-adrenoceptor and the nk- receptor are still expressed by activated cells. we found only the d and d receptors and to a lesser degree the d receptor to be expressed on naïve cd8+ t lymphocytes. all three of them become down-regulated during t cell activation and with the exception of the d receptor these findings are in accordance with watanabe et al.. thus, naïve and activated cd8+ t lymphocytes differ in their level of spontaneous migratory activity. in the naïve state the spontaneous migratory activity is only moderate whereas in the activated state more than half of the cells show migratory activity. this observation is not surprising since the expression pattern of matrix adhesion molecules changes during the activation process and thereby the intracellular signalling. consequently, neurotransmitters have distinct effects on naïve and activated cd8+ t lymphocytes. collagen i receptors such as very late antigen - and vla- are almost absent on naïve cd8+ t cells but become upregulated during activation. this might explain our observation that the naïve lymphocytes show less spontaneous migratory activity than the activated lymphocytes in our migration assay containing 95-98% collagen i as almost single matrix component, with the remainder being comprised of type iii collagen. naïve cd8+ t lymphocytes show an increase in their migratory activity in response to dopamine, norepinephrine and substance p, with dopamine being the strongest inducer. our finding on dopamine is consistent with watanabe et al., who described that dopamine induces chemotaxis in cd45ra+ naïve cd8+ cells via d receptor and postulated a role for endogenous dopamine in the homing of naïve cd8+ t cells. adhesion of ctl to endothelium is selectively regulated by neurotransmitters the ability to extravasate is an important property of immune cells independent of their activation state. naïve lymphocytes need to extravasate during the homing process and activated cells extravasate in order to reach tissue sites of inflammation or injury. the idea that neurotransmitters influence the extravasation process seems likely, because nerve fibres are particularly concentrated around vascular endothelial cells. non-activated cd8+ t lymphocytes show only low adhesion to the endothelium, but a high rolling activity, as we have investigated by our flow-through adhesion assay. in activated cd8+ t lymphocytes, the adhesion significantly increased from ± to ± cells. due to the increased adhesion, the rolling activity is reduced. both adhesion and rolling of activated cd8+ cells is increased by addition of each of the investigated neurotransmitters directly to the experiment. norepinephrine significantly increased the adhesion of the activated cells from ± to ± cells. likewise, the rolling was significantly increased from ± to ± cells/min. dopamine and substance p enhanced the adhesion and rolling of the activated cells only marginally, which was not statistically significant. with regard to naïve, non-activated cd8+ cells, all of the investigated neurotransmitters had only minor effects. however, dopamine significantly increased the adhesion of the naïve, non-activated cells, whereas substance p led to a significant reduction. with regard to dopamine, the finding is in accordance with results from other groups who described an increase in the adhesion of naïve cd45ra+ cd8+ lymphocytes to fibronectin and icam- in response to dopamine but not substance p. this effect was exclusively mediated by the d receptor and led to an activation of the fibronectin receptors vla- and vla-, and the intercellular adhesion molecule - receptor lymphocyte function-associated antigen - on naïve lymphocytes. thus dopamine seems to exhibit a unique function leading to the activation of integrin molecules in naïve cd8+ cells which cannot be induced by norepinephrine or substance p. this observation supports the theory of dopamine being a specific homing factor of naïve cd45ra+ cd8+ cells. since the neurotransmitters were added directly to the cell suspension at the beginning of each experiment both cell types involved - the lympocytes as well as the endothelial cells- are exposed to the substance. interestingly, norepinephrine, but not dopamine or substance p induced a significant increase of the il- release from the endothelial cells, even when pre-activated with the cytokine il-1β. chemokines such as il- are cationic proteins, which bind to heparansulfate and related glycosaminoglycan moieties on the endothelial monolayer, and thereby becomes presented to rolling cells. several studies have conclusively shown that the recruitment of circulating leukocytes at vascular sites in target tissue is linked to activation of gi-protein signalling in leukocytes induced by chemokines presented on the apical site of the endothelial monolayer. these immobilized chemokines are involved in the rapid modulation of the avidity of integrins such as vla- or lfa- expressed by lymphocytes. to confirm this hypothesis, we investigated the surface expression of the according il- receptors cxcr and cxcr on cd8+ lymphocytes: cxcr was almost absent on naïve cells, but it became upregulated during activation. we did not detect an expression of the il- receptor cxcr neither on naïve nor on activated cd8+ t lymphocytes, which corresponds to the results of takata et al. and nishimura et al., but is in conflict with other findings. to investigate a possible role for cxcr in the adhesion of cd8+ t lymphocytes to endothelium in the presence of norepinephrine we blocked this receptor, and cxcr as well, via a specific blocking antibody. this blockade led to a significant reduction of the activated cd8+ t lymphocyte adhesion to the endothelial cells in the presence of norepinephrine compared to the isotype control from ± to ± adherent cells. this reduction was however not completely down to the control level. thus, the effect of norepinephrine on the adhesion of activated cd8+ t lymphocytes shown in fig. 5a is not only mediated by il- instead the effect can be further mediated by other chemokines released by the endothelium or by a direct effect of norepinephrine on the t cells. since we have just screened a subset of chemokines released by endothelial cells, we cannot exclude that the release of further chemokines is affected by norepinephrine. from our experiments, we can exclude that substance p and dopamine as well act in the way as norepinephrine does, since these neurotransmitters do not result in an increased release of il- from the endothelium. degranulation is not influenced by neurotransmitters the effects of the neurotransmitters on the ability of the cells to degranulate were measured by β hexosamidase release in response to cd cross-linking, which mimics the recognition of a target cells without cd co-signalling. this process results in a calcium-dependent release of cytotoxic enzymes from intracellular granules that have been formed during activation. in naïve cd8+ t lymphocytes, the cd3-induced release of β hexosamidase is as high as the release induced by an isotypic control antibody. after activation, the cd3-induced release of β hexosamidase is significantly increased from ± % to ± % release. however, none of the investigated neurotransmitters caused any effect in activated cd8+ t lymphocytes. determination of the calcium-signalling in response to cd cross-linking showed no differences, too. in conclusion, it seems that the investigated neurotransmitters can affect the development of armed effector cells, but when these cells once have been formed, they cannot be inhibited in their effectory function by neurotransmitters.  although we have large and detailed knowledge how certain neurotransmitters act on particular leukocyte subpopulations, the remaining question is still how emotions and sensations are translated into modulatory effects of the immune system, and whether this at all plays a role in the function of the immune system. taken together, we have addressed the question how the neurotransmitters norepinephrine, dopamine and substance p influence the function of cd8+ t lymphocytes. we have identified a regulation pattern showing that neurotransmitters can act either immunostimulatory or immunosuppressive. this depends mainly on the activation state of the t cells. we were able to assign downstream targets of the observed effects. neurotransmitters cannot per se induce new functions in cd8+ t lymphocytes, i.e. migration, adhesion and cytotoxicity, but they play an important role in the fine-tuning of t cell responses. 
therefore, we investigated the influence of norepinephrine, dopamine and substance p on the key tasks of cd8+ t lymphocytes: activation, migration, extravasation and cytotoxicity. in contrast, activated cd8+ t lymphocytes showed a reduced migratory activity in the presence of norepinephrine and substance p. with regard to extravasation we found norepinephrine to induce adhesion of activated cd8+ t cells: norepinephrine increased the interleukin- release from endothelium, which in turn had effect on the activated cxcr1+ cd8+ t cells. furthermore, all of the investigated neurotransmitters increased the spontaneous migratory activity of naïve cd8+ t lymphocytes with dopamine being the strongest inducer. the activation of naïve cd8+ t lymphocytes by cd3/cd cross-linking was inhibited by norepinephrine and dopamine, which was caused by a downregulation of interleukin - expression via erk1/ and nf-κb inhibition. neurotransmitters are specific modulators of cd8+ t lymphocytes not by inducing any new functions, but by fine-tuning their key tasks. neurotransmitters are important regulators of the immune system, with very distinct and varying effects on different leukocyte subsets. so far little is known about the impact of signals mediated by neurotransmitters on the function of cd8+ t lymphocytes.
 neurotransmitters are important regulators of the immune system, with very distinct and varying effects on different leukocyte subsets. so far little is known about the impact of signals mediated by neurotransmitters on the function of cd8+ t lymphocytes. therefore, we investigated the influence of norepinephrine, dopamine and substance p on the key tasks of cd8+ t lymphocytes: activation, migration, extravasation and cytotoxicity.  the activation of naïve cd8+ t lymphocytes by cd3/cd cross-linking was inhibited by norepinephrine and dopamine, which was caused by a downregulation of interleukin - expression via erk1/ and nf-κb inhibition. furthermore, all of the investigated neurotransmitters increased the spontaneous migratory activity of naïve cd8+ t lymphocytes with dopamine being the strongest inducer. in contrast, activated cd8+ t lymphocytes showed a reduced migratory activity in the presence of norepinephrine and substance p. with regard to extravasation we found norepinephrine to induce adhesion of activated cd8+ t cells: norepinephrine increased the interleukin- release from endothelium, which in turn had effect on the activated cxcr1+ cd8+ t cells. at last, release of cytotoxic granules from activated cells in response to cd cross-linking was not influenced by any of the investigated neurotransmitters, as we have analyzed by measuring the β-hexosamidase release.  neurotransmitters are specific modulators of cd8+ t lymphocytes not by inducing any new functions, but by fine-tuning their key tasks. the effect can be either stimulatory or suppressive depending on the activation status of the cells. 