Download Norepinephrine Inhibits Energy Metabolism of Human

Bioscience Reports, Vol. 21, No. 5, October 2001 ( 2002)
Norepinephrine Inhibits Energy Metabolism of
Human Peripheral Blood Mononuclear Cells
via Adrenergic Receptors
Jan D. Lünemann,1 Frank Buttgereit,2 Robert Tripmacher,2
Christoph G. O. Baerwald,3 Gerd-Rüdiger Burmester,2 and Andreas Krause4
Receiûed May 18, 2001
Previous studies demonstrated that the adaptive response to stressors and inflammatory
signals involves the activation of the automotic nervous system. Catecholamines have been
shown to modulate the activity of various immune effector cells directly via membrane
adrenergic receptors. Here, we investigated immediate effects of norepinephrine on energy
metabolism of immune cells. Norepinephrine inhibits oxygen consumption of human peripheral blood mononuclear cells at concentrations that are relevant to its physiological
range. The β -adrenoreceptor antagonist propranolol, but not the α -adrenoreceptor antagonist phentolamine reversed the norepinephrine induced inhibition in quiescent cells. Conversely, phentolamine but not propranolol is capable of blocking norepinephrine mediated
effects in mitogen activated human peripheral blood mononuclear cells. Our data indicate
that the sensitization of α - and β -adrenoreceptors on immune cells is differentially regulated, and that these processes depend on the activation state of these cells. These findings
have important implications for the understanding of stress-induced suppression of immune
function and may contribute to the elucidation of the pathogenesis of immunologically
mediated diseases.
KEY WORDS: Adrenergic receptor; blood cell; catecholamine; energy metabolism;
norepinephrine.
INTRODUCTION
Since the identification of adrenergic receptors on lymphocytes, monocytes兾macrophages and granulocytes, strong evidence has accumulated demonstrating that the
sympathetic nervous system (SNS) features important immunoregulatory properties
(Ader et al., 1990). Engagement of adrenergic receptors was shown to affect lymphocyte activation, cytokine production, proliferation, and functional activity of different lymphoid cells (Elenkov et al., 2000).
1
Department of Neurology, Division of Neuroimmunology, Charité University Hospital, Berlin,
Germany.
2
Department of Medicine, Rheumatology and Clinical Immunology, Charité University Hospital, Berlin,
Germany.
3
Department of Rheumatology, University Hospital, Leipzig, Germany.
4
To whom correspondence should be addressed. E-mail: [email protected]
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0144-8463兾01兾1000-0627兾0  2002 Plenum Publishing Corporation
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Lünemann et al.
The SNS and the immune system are anatomically linked by extensive noradrenergic and peptidergic intraparenchymal innervation of primary and secondary
lymphoid organs (Felten et al., 1987). In the periphery, norepinephrine (NE) is also
released nonsynaptically, i.e., from free nerve endings into a large extraneural space
(Vizi, 2000). Hence, the neurotransmitter diffuses a considerable distance before
interacting with its specific membrane receptor on target cells. In contrast to rapid
and phasic synaptic interactions, this nonsynaptic neutrotransmission appears to act
relatively slowly (within minutes) and exhibits more tonic effects, that are conceivable of fine-tuning initial steps of immune responses (Vizi, 1998).
Most of the previous in ûitro studies investigating the immunomodulatory
effects of catecholamines focussed on lymphocyte activation, proliferation, and differentiation as comparatively long-ranging consequences of an initially short-term
interaction. Relatively little is known about the immediate effects of catecholamines
on immune responses that may be part of adaptive changes during acute stress.
To reflect rapid catecholamine action, we applied an in ûitro model that has
been used successfully in several different systems and tested for validity in various
studies (Buttgereit et al., 1993; 1994; 1994b; Schmid et al., 2000). We investigated
whether NE exhibits immediate effects on oxygen-consumption of quiescent and
Concanavalin A (Con A) activated human immune cells, namely peripheral blood
mononuclear cells (PBMC ). Oxygen-consumption clearly reflects the activity of processes that ensure proper functions of immune cells, since their respiration rate can
be quantitatively linked to energy metabolism (Buttgereit et al., 1992; 2000). Thus,
NE induced alterations in oxygen metabolism mirror changes in ATP-producing
and兾or ATP-consuming pathways which are highly relevant for subsequent cellular
immune processes.
Exposure of quiescent cells to mitogenic lectins such as Con A triggers a prototypical range of biochemical changes that initiate, or coincide with the metabolic
depression of quiescent cells in the G0 phase (Krauss et al., 1999). The principle
changes are similar to those that are likely to be involved in the pathogenesis of
immunological mediated diseases (Buttgereit et al., 1992).
Here, we report that NE inhibits the respiration rate of quiescent and Con A
activated human PBMC within seconds. The β -adrenoreceptor antagonist propranolol, but not the α-adrenoreceptor antagonist phentolamine reversed the NE induced
inhibition in quiescent cells. Conversely, phentolamine but not propranolol is capable of blocking NE mediated effects in mitogen activated human PBMC. Our data
indicate that catecholamines inhibit energy metabolism of quiescent and activated
immune cells. This effect is mediated by differentially regulated membrane α - and
β -adrenergic receptors depending on the activation status of these cells.
MATERIAL AND METHODS
Preparation and Incubation of Cells
PBMC were prepared from healthy blood donors using a standard method
based on Ficoll-Hypaque centrifugation. In detail, venous blood was diluted with
saline and density gradient centrifugation was performed at 400g for 20 min. The
Norepinephrine in PBMC
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PBMC enriched interphase was isolated, washed with saline and resuspended at
2B107 cells兾ml. Achieving high cell concentrations is necessary to get reliable and
reproducible results in oxygen consumption determined by using a Clark electrode.
The incubation medium was a 1 :1 mixture of Eagle’s Basal Medium with a Hanks’
salt mixture supplemented with 0.15 M Tris兾HCl at pH 7.4 and with 19 L-amino
acids each at 0.2 mM final concentrations, but lacking glutamine and NaHCO3. The
medium was filtered through a 0.2 µm-pore-size filter to remove undissolved particles
as previously described (Buttgereit et al., 1992; 1993). Cells were stored and incubated for up to three hours in wide plastic flasks to ensure aeration at 4°C. The
PBMC consisted of 85–95% lymphocytes and 5–15% monocytes, as determined by
FACS-analysis. Trypan blue staining revealed the viability of freshly isolated cells
to be greater than 95%.
Measurements of Respiration Rate
Oxygen consumption was measured amperometrically in a 0.7 ml aliquot of cell
suspension with a Clark electrode for up to 15 min as described previously (Buttgereit et al., 1992, 1993, 1994). The cell suspensions were magnetically stirred in a
Perspex incubation chamber of the electrode and thermostatically maintained at
37°C. NE (Sigma-Aldrich, Deisenhofen, Germany) was dissolved in the medium
mixture mentioned above and added to the cell suspensions at final concentrations
of 10−5 M, 10−7 M and 10−9 M. In a second approach, cells were preexposed to the
adrenergic antagonists propranolol (Dociton, Zeneka, Plankstadt, Germany) and
phentolamine (Sigma-Aldrich, Deisenhofen, Germany) at a final concentration of
10−3 M. For mitogen activation experiments Concanavalin A (Sigma-Aldrich,
Deisenhofen, Germany) was dissolved in water and added to cell suspensions at
75 µg兾ml. In previous investigations (Schmid et al., 2000) this concentration of Con
A was shown to produce a standardized and reproducible cell stimulation that is
reflected by a significant increase of respiration persisting for at least ten minutes
(the time at which all oxygen is removed from the chamber due to respiration of the
cells). At first glance this concentration appears to be rather high, but we have found
the Con A effect on respiration also depended on the cell number (not documented).
The cell concentration we necessarily use in our experiments is about 5–40 times
higher than that used by other investigators who add Con A only at 1–5–20 µg兾ml.
Therefore, related to the cell number our Con A concentration corresponds to
2.5 µg兾106 cells which is within the same range as reported by other authors.
Respiration rates were measured during 1–3 min to evaluate the basal respiration rate (untreated cells) and another 1–3 min after addition of each reagent. The
maximal effect was achieved within seconds and was constant thereafter.
Statistics
Analyses were performed using the paired Student t-test. Calculation was carried out using SPSS 10.0 software for Windows (SPSS Inc., Chicago, IL, USA). A
p value of F0.05 was regarded as significant.
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RESULTS
1. Norepinephrine inhibits oxygen consumption in quiescent and Con A activated human PBMC.
Aerated, quiescent human PBMC were placed into the incubation chamber and
the respiration rate was measured as mentioned above. After respiration had reached
a steady state of 6.91J0.13 nmol O2兾minB107 cells (meanJSEM for 72 cell preparations), the cells were exposed to serial dilutions of NE. As shown in Fig. 1a, NE
inhibited oxygen consumption of PBMC in a dose-dependent manner. The maximal
inhibitory effect of NE was achieved with 10−5 M concentrations, although a concentration of 10−9 M already showed a significant inhibition of respiration in quiescent
PBMC ( pH0.001).
In a second approach, the immune cells were stimulated with Con A at a
final concentration of 75 µg兾ml. The stimulation caused an almost twofold increase
of oxygen consumption within seconds leading to a respiration rate of
10.82J1.05 nmol O2兾minB107 cells (meanJSEM for 26 cell preparations; pH0.001
as compared to baseline values). Figure 2a shows the data for human PBMC that
were incubated in medium with Con A alone (control) or with Con A plus various
concentration of NE. NE was found to inhibit the Con A induced stimulation of
oxygen consumption in a dose-dependent manner ( pH0.05 for NE concentrations
of 10−5 M).
2. Propranolol, but not phentolamine blocks NE induced inhibition of respiration in quiescent PBMC.
To determine whether the effects observed were mediated via specific
α - or β -adrenergic receptors, the β -adrenoreceptor antagonist propranolol and the
α -adrenoreceptor antagonist phentolamine were added to quiescent PBMC at final
concentrations of 10−3 M. The antagonists did not exert any significant effect on the
respiration rate by themselves ( pH0.05, data not shown). As demonstrated in Fig.
1b, preexposure to propranolol significantly reversed the inhibitory effect of NE at
all three concentrations tested ( pH0.01). Although there was a trend towards a
reduction of the NE induced inhibition by preincubation with phentolamine in
higher concentrations as well, these values did not reach significance ( pF0.05).
Thus, in our hands phentolamine was not capable of blocking the action of NE on
quiescent PBMC.
3. Phentolamine, but not propranolol blocks NE induced inhibition of respiration in Con A activated PBMC.
To evaluate whether the inhibitory effect of NE in mitogen activated cells was also
mediated via adrenergic β -receptors, further experiments were performed with Con A
stimulated PBMC. Again, NE inhibited the Con A induced cell stimulation. In contrast to quiescent PBMC, propranolol failed to antagonize the effect of NE in mitogen
stimulated PBMC. However, NE induced inhibition of Con A stimulated oxygenconsumption could partially be reversed by preexposure to the α -adrenoreceptor
blocker phentolamine (Fig. 2b). Moreover, the results showed that in some experiments the adrenoreceptor antagonists displayed a tendency towards a stimulation
Norepinephrine in PBMC
Fig. 1. Norepinephrine inhibits oxygen consumption in quiescent PBMC via
β-adrenergic receptors. Human PBMC were incubated in medium, followed
by application of serial dilutions of NE. Respiration rate was assayed by a
Clark electrode as described in the Methods section. NE significantly
decreases oxygen consumption in a dose-dependent manner (*pF0.05 for all
three concentrations tested). Preexposure of quiescent cells with the β-adrenergic antagonist propranolol, but not with the α-adrenergic antagonist phentolamine almost completely reversed the NE induced inhibition of oxygenconsumption (§ pF0.05 for propanolol vs. no antagonist). Results are
meansJS.E. for 26 cell preparations with NE only (Fig. 1a) and ten experiments with above mentioned antagonists (Fig. 1b).
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Fig. 2. The inhibitory effects of norephrine on oxygen consumption in
mitogen activated PBMC is mediated via α-adrenergic receptors. PBMC were
incubated in medium and activated by 75 µg兾ml Con A. Addition of NE led
to a significant dose-dependent decrease of oxygen-consumption (*pF0.05
for NE 10−5 M, Fig. 2a). Pretreatment of Con A activated cells with phentolamine completely reversed the NE induced inhibition (§ pF0.05 for
Con ACNE vs. phentolamineCCon ACNE), whereas propranolol failed to
display any effect (Fig. 2b). Results are meansJS.E. for 26 cell preparations
with Con A and NE only and ten blocking experiments.
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of oxygen-consumption additional to Con A, but these effects turned out not to be
significant ( pH0.05).
DISCUSSION
Evidence accumulated over the last two decades indicating that the autonomic
nervous system influences the immune response by various pathways including
activation and modulation of membrane adrenergic receptors on immunocompetent
cells. However, data concerning the mechanisms of action and effects of catecholamines on immune cells are still controversial and incompletely understood. Previous studies revealed that the time point of adrenergic stimulation during the course
of an immune response and thus, the activation status of the immune cells clearly
influence the net effect of catecholamine action (Deplechin and Letesson, 1981). To
further investigate immunomodulatory properties of catecholamines, we applied a
well established in ûitro model that allowed us to determine immediate effects of NE
on immune cells by measurement of oxygen consumption.
The present study demonstrates that NE is capable of decreasing oxygen consumption in human quiescent and mitogen activated PBMC within seconds in a
dose-dependent manner. Importantly, in quiescent cells NE was shown to inhibit
oxygen consumption even at nanomolar levels that are comparable to its physiological concentration in peripheral blood.
In our test system we have used an incubation medium where glycolysis is negligible (Buttgereit et al., 1993; 1994). Therefore, cellular oxygen consumption is a
direct measure to quantificate energy metabolism in PBMC, since oxidation of fuel
molecules to drive oxidative phosphorylation represents the major energy source
and ATP is the principal donor of free energy (Saraste, 1999). Immunocompetent
cells require considerable amounts of energy to maintain housekeeping functions
and to exert specialized immune functions. The pattern of energy consuming processes has been investigated in detail for lymphocytes. In quiescent thymocytes, most
of the mitochondrial oxygen consumption among the identified ATP consuming
pathways is used for protein synthesis and for transport of cations by the NaC兾KC
ATPase. As demonstrated in our study, Con A stimulation leads to an almost twofold increase of oxygen consumption within seconds, reflecting higher ATP production to balance a greater demand of specific activated processes that involve
additional RNA兾DNA synthesis and recruitment of the Ca2C-ATPase (Buttgereit et
al., 2000). While short term downregulation of energy consuming immune functions
appears to be part of a physiologic stress-response, prolonged stress situations might
lead to an impairment of immunologically relevant processes with pathophysiological consequences. Energy depletion in human immunocytes greatly inhibits their
activation and subsequent effector functions such as antigen-processing兾presentation, production of and receptor affinity to cytokines, synthesis of effector molecules,
performing cytotoxicity and regulatory properties (Buttgereit et al., 2000). Therefore, NE induced changes in energy metabolism might affect processes of crucial
importance for the efficacy of subsequent specific immune functions.
Blocking experiments with propranolol showed that in unstimulated PBMC the
inhibitory effect of NE was mediated via β -adrenoreceptors only. Conversely, the
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action of NE on respiration of lectin activated immune cells could be reversed solely
by the α -adrenoreceptor antagonist phentolamine.
While ligand binding studies and functional experiments revealed the presence
of high-affinity membrane β -adrenoreceptors, mainly of the β 2 subclass, on human
peripheral lymphocytes and other hematopoietic cells (Madden et al., 1995), the
expression and functional role of α -adrenoreceptors on immune cells is still a matter
of debate. Albeit Casale et al. (Casale and Kaliner, 1984) did not detect α 1- or α 2adrenoreceptors on human mononuclear blood cells by radioligand binding analyzes, functional studies indicated that α -adrenoreceptors are capable of modulating
immune responses under certain circumstances (Elenkov et al., 2000).
Based on early in ûitro studies, a simple dichotomy was established between
α - and β -adrenoreceptor mediated immunoregulation. β -adrenergic stimulation was
shown to inhibit such activities as lymphocyte proliferation, antibody-secretion, and
synthesis of pro-inflammatory cytokines, whereas α -adrenoreceptors were considered to mediate opposite, i.e. more stimulatory effects on lymphocytes (Bourne
et al., 1974). Recent data drew a more complex scenario of catecholamine mediated
immunoregulation and indicated that the functional signals mediated via α -receptors appear to be significant in chronic inflammatory diseases only. Coderre and
colleagues (Coderre et al., 1991) reported that α 2-adrenoreceptor stimulation in ûiûo
was capable of suppressing experimental arthritis in the rat and Heijnen et al.
(Heijnen et al. 1996) demonstrated that in contrast to normal blood volunteers
young patients with polyarticular juvenile rheumatoid arthritis display functional
α 1-adrenoreceptors on peripheral blood leukocytes. Further studies revealed that at
least in patients with rheumatoid arthritis the expression of α -adrenoreceptors on
PBMC depends on the systemic disease activity. Catecholaminergic effects mediated
via α -adrenoreceptors were detectable in cells from patients with high disease
activity only (Wahle et al., 1999).
Using lectin activated human PBMC, our data demonstrate the presence of
α -adrenoreceptors on these cells and the immunosuppressive property of α -adrenoreceptor mediated signaling under these in ûitro conditions, that are likely to be
similar to pathophysiological processes in ûiûo.
Membrane adrenoreceptors directly activate G-proteins that regulate the transduction of transmembrane signals from the cell surface to a variety of intracellular
effectors. The mitogen stimulation obviously led to a desensitization of β -receptors,
but to a sensitization of a α -receptors within three minutes only. Adrenoreceptor
function is tightly regulated by rapid processes including (de)coupling to G-proteins
within seconds and internalization or externalization of the receptor itself within
minutes (Roth et al., 1991, Barnes, 1995) respectively. Long-term up- or down-regulation of these receptors requires a timeframe of several hours. Therefore, our data
indicate that mitogen stimulation directly influences rapid adrenoreceptor modulation and signaling. However, the precise mapping and type of adrenoreceptor
expression on different lymphoid cells and their coupling to intracellular pathways
in dependence of their activation status clearly need further studies.
Measuring oxygen consumption as a determinant of energy metabolism serves
as an excellent in ûitro model to investigate immediate effects of neurotransmitters
and hormones on immunocompetent cells. This model may therefore help to further
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unravel the impact of acute stress responses on immune functions. Moreover, since
investigations on immunologically mediated diseases like rheumatoid arthritis
(reviewed in Baerwald et al., 2000), multiple sclerosis (reviewed in Schorr et al.,
1999) and their corresponding animal models, revealed an important influence of
the SNS on the immune response in these autoimmune diseases, the elucidation of
bioenergetic processes of immunocompetent cells affected by catecholamines appears
to be of vital relevance for understanding their pathogenesis. Therefore, characterization of catecholamine induced effects on energy metabolism of immune cells may
help to identify new promising targets for future therapeutic strategies.
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