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HUMAN LYMPHOCYTE ß 2 - ADRENOCEPTORS
AND IMMUNE FUNCTION
HUMAN LYMPHOCYTE ß2-ADRENOCEPTORS
AND IMMUNE FUNCTION
Een wetenschappelijke proeve op het gebied
van de Medische Wetenschappen,
in het bijzonder de Geneeskunde
PROEFSCHRIFT
TER VERKRIJGING VAN DE GRAAD VAN DOCTOR AAN DE
KATHOLIEKE UNIVERSITEIT NIJMEGEN,
VOLGENS BESLUIT VAN HET COLLEGE VAN DECANEN
IN HET OPENBAAR TE VERDEDIGEN OP
VRIJDAG 18 SEPTEMBER 1992 DES NAMIDDAGS TE 1.30 UUR PRECIES
DOOR
LAMBERTUS JOHANNES HUBERTUS VAN TITS
GEBOREN OP 28 OKTOBER 19 61
TE BOXMEER
1992
Druk: Krips Repro Meppel
Promotores: Prof. Dr. О.-E. Brodde, Universität der
Gesamthochschule Essen
Prof. Dr. Th. Thien
Co-promotor: Dr. S. Graafsma
ISBN 90-9005301-8
The studies presented in this thesis were performed in the
Department of Medicine, Division of Internal Medicine, Biochemical
Research Laboratories, University of Essen, Essen, Germany.
Financial support by the Netherlands Heart Foundation for the
publication of this thesis is gratefully acknowledged.
voor mijn ouders
voor Elly
CONTENTS
GENERAL INTRODUCTION 1
CHAPTER 1. Agonist-specific and non-specific in vitro desensitization of human lymphocyte ß2-adrenoceptors ... 11
CHAPTER 2. Catecholamines increase lymphocyte ß2-adren-ergic receptors via a ß2-adrenergic, spleen-dependent process 31
CHAPTER 3. Effects of insulin-induced hypoglycemia on ßj-adrenoceptor density and proliferative responses of human lymphocytes 62
CHAPTER 4. Cyclic AMP counteracts mitogen-induced inositol phosphate generation and increases in intracellular Ca2+-concentrations in human lymphocytes 79
CHAPTER 5. Adrenergic involvement in control of lymphocyte immune function 103
CHAPTER 6. Reduced proliferation of human lymphocytes after infusion of isoprenaline is unrelated to impairments in transmembrane signalling 118
SUMMARY 132
SAMENVATTING 139
DANKSAGUNG 144
LIST OF PUBLICATIONS 146
CURRICULUM VITAE 149
GENERAL INTRODUCTION
This thesis deals with effects of catecholamines on human
lymphocyte ß2-adrenergic receptors and on several parameters of
lymphocytes concerning immune function. The following section
gives a concise description of the sympathetic nervous system
and of the lymphocytes. Special attention is given to the
adrenergic receptors, the structures playing a crucial role in
signal transmission of the sympathetic nervous system. The chapter
is concluded with a summing up of questions that will be addressed.
THE SYMPATHETIC NERVOUS SYSTEM
Visceral functions of the body are controlled by the autonomic
nervous system, also called the involuntary nervous system,
referring to the fact that many of the activities take place at
the unconscious level. Its primary function is to help to maintain
a stable internal environment in the body against those forces
that tend to alter it. The autonomic nervous system is represented
in both the central and peripheral nervous system. Signals are
transmitted to different organ systems of the body through two
major subdivisions called the fortho-; sympathetic (1) and
parasympathetic system. In general, the sympathetic system
stimulates those activities which are most dramatically expressed
and mobilized during emergency and stress situations, popularly
called the "fight, fright, and flight activities". In contrast,
the parasympathetic system stimulates those activities that are
associated with the conservation and restoration of the energy
stores. Together the two systems function in concert to maintain
the cellular activities at a level proportional to the intensity
of the stress situation and the emotional state of the individual.
Both the sympathetic and parasympathetic system consist of
preganglionic and postganglionic neurons. Messages transported
along the nervous fibres (pre- as well as postganglionic) are
finally translated into the release of a neurotransmitter at the
1
nerve endings. Whereas the neurotransmitter substance acetyl
choline is released at each synaptic junction between pre- and
postganglionic neurons, the nature of the neurotransmitter
substances released by the postganglionic neurons of the two
systems is different. The neurotransmitter substance at the
parasympathetic postganglionic effector junction is acetylcholine
and the system is referred to as a "cholinergic system". The
postganglionic nerve endings of the sympathetic nervous system
usually secrete norepinephrine, one of the catecholamines, and
are said to be noradrenergic (derived from noradrenaline, the
British name for norepinephrine). Norepinephrine is synthesized
from the amino acid tyrosine by the following biochemical pathway:
tyrosine — > dopa (dihydroxyphenylalanine) — > dopamine — >
norepinephrine. Synthesis of norepinephrine begins in the axoplasm
of the nerve endings, but dopamine is transported into the
norepinephrine storage vesicles, where norepinephrine synthesis
takes place.
An action potential at the sympathetic postganglionic nerve
terminal results in release of norepinephrine into the synaptic
cleft. The released norepinephrine within the synaptic cleft has
several fates. Most of it is inactivated by re-uptake into the
noradrenergic nerve terminals themselves through active trans
port. Then there is diffusion away from the nerve endings into
the surrounding body fluids. Furthermore there is metabolic
degradation by monoamine oxidase (intraneuronally) and by
catechol-O-methyl transferase (extracellularly). Essential for
continuing transport of the signal, however, is binding of
norepinephrine with receptor sites on the postsynaptic membrane.
In general, the receptor-site is a protein molecule located in
the cell membrane. Binding of the neurotransmitter with its
receptor causes a basic change in the structure of the protein
molecule. Transmembrane signalling then occurs via a change in
the activity of the effector, usually an ion channel or an enzyme
in the cell membrane. Interaction of receptor and effector is
accomplished by coupling proteins which are regulated by guanine
nucleotides such as guanosine triphosphate.
2
ADRENOCEPTORS
The receptors that bind norepinephrine are called adrenergic
receptors or adrenoceptors, derived from adrenaline (the British
name for epinephrine, the catecholamine that is formed by
N-methylation of norepinephrine and that mimics the action of
norepinephrine). On effector cells innervated by the sympathetic
nervous system, two types of chemically defined adrenoceptors
exist: alpha (a) and beta (ß) adrenoceptors. This subdivision is
based on different relative rank orders of potency of several
catecholamines for stimulating a variety of physiological effects
in different organ systems (2). α-Adrenoceptors have a higher
affinity for epinephrine and norepinephrine than for isoprenaline,
a synthetic catecholamine. ß-Adrenoceptors, on the other hand,
have a higher affinity for isoprenaline than for epinephrine and
norepinephrine. According to differences in rank order of potency
of agonists and antagonists, subtypes of a- (3,4) and ß-adre-
noceptors (5,6) are defined. a^^-Adrenoceptors cause changes in
the concentration of intracellular calcium, which turns on the
highly complex calcium regulatory system. aj-Adrenoceptors are
inhibitorily coupled to the membrane-bound enzyme adenylate
cyclase, which upon stimulation generates the second messenger
3'5'-cyclic adenosine monophosphate. ß-Adrenoceptors are exci-
tatorily coupled to the adenylate cyclase (Figure 1).
ATP cAMP
Figure 1. Schematic representation of adrenoceptor transmembrane signalling. Rg, stimulatory receptor; RL, inhibitory receptor; Gs, stimulatory G-protein; CL, inhibitory G-protein; AC, adenylate cyclase; ATP, adenosine triphosphate; CAMP, cyclic adenosine monophosphate.
3
Activation of effector cells by the sympathetic nervous
system occurs either directly or indirectly (7). The indirect
effects are accomplished via stimulation of the adrenal glands.
The adrenal medullae are controlled by the preganglionic cho
linergic fibers of the sympathetic system (8). Upon sympathetic
stimulation the adrenal gland secretes large guantities of
epinephrine and norepinephrine (10-20%) into the circulating
blood. Norepinephrine is produced in one type of cell in the
adrenal gland by the same pathway as described above. Epinephrine
is formed in another type of cell by the methylation of nore
pinephrine. In this respect these catecholamines act as hormones.
They are carried via the blood to all tissues of the body and
have almost the same effects on the different organs as those
caused by direct sympathetic stimulation. However, whereas the
norepinephrine secreted directly in a tissue by noradrenergic
nerve endings remains active for only a few seconds, the effects
of the humoral catecholamines are much more sustained because
they are deactivated relatively slowly.
Most organs innervated by the autonomic nervous system are
dominantly controlled by either the sympathetic or the para
sympathetic nervous system. Regulation of the activity of the
organs occurs by decreasing or increasing either the sympathetic
or the parasympathetic drive. Due to the existence of a basal
tone, a change in the degree of autonomic stimulation can lead
to a change in the activity of the organ in two directions, i.e.
a decreased or an increased effect. The overall sympathetic tone
results from basal secretion of epinephrine and norepinephrine
by the adrenal medullae, in addition to the tone resulting from
direct sympathetic stimulation. Apart from variation in the degree
of stimulation, the activity of the organs is believed also to
depend on the density of the receptors present on the organs.
The concentration of the adrenergic receptors on cells is not
fixed but dynamically regulated by a wide variety of physiological
and pathophysiological variables and may be either increased or
decreased in different conditions. Patients suffering from
diseases such as asthma, hypertension, congestive heart failure,
4
ischemie heart disease and thyroid disease have been shown to
display altered adrenergic receptor densities on tissues involved
in the disease (9,10). An altered adrenergic receptor concen
tration may contribute to or determine an altered tissue
sensitivity to catecholamine effects. The sympathetic nervous
system innervates three types of effector cells: smooth muscle
cells, cardiac muscle cells, and glandular cells. Thus, adrenergic
receptors are widely distributed in humans. They exist for example
on organs of the respiratory, the circulatory, the digestive,
the urinary and the reproductive systems, but also in the brain,
on adipocytes, and on blood cells.
LYMPHOCYTES
Peripheral blood lymphocytes contain a homogeneous population
of ß2-adrenergic receptors (11) excitatorily coupled to the
adenylate cyclase, and, since they are readily accessible, provide
a convenient source of material for study of the adrenergic
receptors (12). Most of our knowledge of the human ß-adrenergic
receptor-effector system has emerged from studies on the lym
phocyte ß2-adrenergic receptor. However, relatively little
attention has been given to the significance of the system for
lymphocyte function.
Lymphocytes are part of the immune system, together with
the macrophages they play a role in acquired immunity. The cells
develop in primary lymphoid organs. There are two major primary
lymphoid organs, the thymus gland in which Τ lymphocytes (thy-
mus-derived) develop, and the bursa of Fabricius (avian) or its
equivalent the bone marrow (mammals), in which В lymphocytes
(bursa-derived) develop. Proliferation and differentiation occurs
in secondary lymphoid organs such as the spleen, the lymph nodes
and the tonsils. В lymphocytes have a humoral immune function:
they synthesize and secrete antibodies. Τ lymphocytes have a
cellular immunomodulatory function at various levels of the immune
response. According to their most prominent function, three
subclasses of Τ cells can be distinguished. Helper Τ cells serve
a stimulatory immunomodulatory function: they help В cells to
5
produce antibodies and regulate many aspects of the immune response
by the release of lymphokines. They can also help to generate
cytotoxic Τ cells. Cytotoxic Τ cells are effector cells with an
important role in the immune reaction against intracellular
parasites and viruses. Suppressor Τ cells serve a suppressive
immunomodulatory function in the way that they suppress the
function of the helper Τ cells and of the В cells. In addition,
another subclass of lymphocytes, the natural killer (NK) cells,
has a cytotoxic function. The NK cells recognize cell surface
changes that occur on some virally infected cells and some tumour
cells. They bind to these target cells and kill them.
The existence of subtype-specific markers on the surface of
the lymphocytes enables identification of the functionally
different subclasses of lymphocytes (13). Furthermore, there are
several naturally occurring substances that are mitogenic for,
i.e. have the capacity to bind to, and to trigger proliferation
and differentiation of, many clones of lymphocytes in a way
similar to the antigenic activation of lymphocytes in vivo (14).
For example, two plant glycoproteins (lectins), concanavalin A
and phytohemagglutinin, are potent mitogens for Τ cells and are
useful in the identification and study of the function of this
class of cells. Pokeweed mitogen is considered to be mitogenic
for both Τ cells and В cells. The lectins interact with specific
receptors on the lymphocytes. These receptors couple to the
phospholipase C/inositoltriphosphate (IP3)/diacylglycerol (DAG)
pathway (15). Activation of the receptor results in the phos
pholipase C-mediated hydrolysis of phosphatidylinositol-bis-
phosphate into IP3 and DAG, molecules which both have second
messenger functions (16). IP3 mobilizes intracellular Ca2+, and
DAG activates protein kinase С (Figure 2) . Similar to the adenylate
cyclase/cyclic AMP pathway, also in this signalling pathway, the
receptors seem to be coupled to the effector by G-proteins (17-19) .
It is generally accepted that the immune system can be
modulated by endocrine and nervous factors, including the sym
pathetic nervous system (20). Moreover, stress is increasingly
б
PIP, ІРЭ + DAG
Figure 2. Schematic representation of phospholipage C/inositoltriphosphate
(IP3)/diacylglycerol (DAG) transmembrane signalling. R, receptor; G, G-pro-
tein; PLC, phospholipase С; PIP2» phosphatidylinositol-bisphosphate.
reported in association with immunosuppression (21) . On the other
hand, lymphocyte ß2-adrenoceptors are very sensitive to acute
conditions of stress (22-26). The adaptation which occurs, an
increase in ß2-adrenoceptors, has thus far only been observed in
lymphocytes and might be lymphocyte-specific. Thus, lymphocytes
are an interesting model to investigate the relation between the
sympathetic nervous system and the immune system.
AIMS OF THE STUDY
Studies were designed to discover the mechanism underlying
the rapid increases of lymphocyte ß2-adrenoceptors in vivo
following acute catecholamine exposure. A standardized method of
infusion of isoprenaline into healthy volunteers was used to
provoke the increases in ß2-adrenoceptors on peripheral lym
phocytes. A panning technique for preparation of purified lym
phocyte subsets was developed so that ß2-adrenoceptor densities
could be measured not only on non-fractionated lymphocytes but
also on separate subsets. To evaluate effects of activation of
the sympathetic nervous system on lymphocyte immune function, in
addition to measurements of ß2-adrenoceptor density, the subset
composition of peripheral blood mononuclear leucocytes and
lymphocyte in vitro proliferative responses to various mitogenic
agents were determined. In search for a biochemical point of
contact, also in vitro stimulation of lymphocyte ß2-adrenoceptors
7
was investigated in relation to lymphocyte immune function. For
this purpose mitogenic agents were used to mimic the activation
of resting lymphocytes by antigens, and formation of inositol
phosphates and increases in intracellular calcium were determined.
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1. Cryer P.E., 1980. Physiology and pathophysiology of the human
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4. Starke Κ. , 1981. α-Adrenoceptor subclassification. Rev.
Physiol. Biochem. Pharmacol. 88, 199-236.
5. Lands A.M., Arnold A, McAuliff JP, Luduena FP and Brown TG,
1967. Differentiation of receptor systems activated by sympa
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1978. Norepinephrine: hormone and neurotransmitter in man. Am.
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8. Shah S.D., Tse T.F., Clutter W.E. and Cryer P.E., 1984. The
human sympathochromaffin system. Am. J. Physiol. 247, E380-E384.
9. Lefkowitz R.J., Caron M.G., Stiles G.L., 1984. Mechanisms of
membrane-receptor regulation. Biochemical, physiological, and
clinical insights derived from studies of the adrenergic
receptors. N. Engl. J. Med. 310, 1570-1579.
10. Stiles G.L., Caron M.G. and Lefkowitz R.J. , 1984. ß-Adrenergic
receptors: biochemical mechanisms of physiological regulation.
Physiol. Rev. 64, 661-743.
11. Brodde O.-E., Engel G., Hoyer D. , Bock K.D. and Weber F.,
1981. The ß-adrenergic receptor in human lymphocytes: subclas
sification by the use of a new radio-ligand,
(±)-125iodocyanopindolol. Life Sciences 29, 2189-2198.
8
12. Motulsky H.J., Insel P.A., 1982. Adrenergic receptors in man.
Direct identification, physiologic regulation and clinical
alterations. N. Engl. J. Med. 307, 18-29.
13. Horejsi V. and Bazil V., 1988. Surface proteins and glyco
proteins of human leucocytes. Biochem. J. 253, 1-26.
14. Janossy G. and Greaves M.F., 1971. Lymphocyte activation.
Response of Τ and В lymphocytes to phytomitogens. Clin. Exp.
Immunol. 9, 483-498.
15. Nordin A.A. and Proust J.J., 1987. Signal transduction
mechanisms in the immune system. Endocrinology and Metabolism
Clinics 16, 919-945.
16. Berridge M.J., 1987. Inositol trisphosphate and diacylg-
lycerol: two interacting second messengers. A. Rev. Biochem. 56,
159-193.
17. Cockcroft S. and Stutchfield J., 1988. G-proteins, the
inositol lipid signalling pathway, and secretion. Phil. Trans.
R. Soc. Lond. В 320, 247-265.
18. Harnett M.M. and Klaus G.B., 1988. G protein regulation of
receptor signalling. Immunol. Today. 9, 315-320.
19. Lo W.W.Y. and Hughes J., 1987. Receptor-phosphoinositidase
С coupling. Multiple G-proteins? FEBS Letters 224, 1-3.
20. Dunn A.J., 1988. Nervous system-immune system interactions:
an overview. J. Receptor Res. 8, 589-607.
21. Khansari D.N., Murgo A.J. and Faith R.E., 1990. Effects of
stress on the immune system. Immunol. Today. 11, 170-175.
22. Butler J., Kelly J.G., O'Malley K. and Pidgeon F., 1983.
ß-Adrenoceptor adaptation to acute exercise. J. Physiol. Lond.
344, 113-118.
23. Brodde O.-E., Daul Α., and O'Hara N., 1984. ß-Adrenoceptor
changes in human lymphocytes, induced by dynamic exercise.
Naunyn-Schmiedeberg's Arch. Pharmacol. 325, 190-192 (Erratum:
Naunyn Schmiedeberg's Arch. Pharmacol. 328, 362, 1985) .
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Latham Κ., 1985. Beta receptors in peripheral mononuclear cells
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9
M., DiLallo M., Smith D.A. , Lai C.-Y.C, Motulsky H. J. , 1986. In
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26. Graafsma S.J., Van Tits В., Westerhof В., Van Valderen R.,
Lenders J.W.M., Rodrigues de Miranda J.F., Thien Th., 1987.
Adrenoceptor density on human blood cells and plasma catecho
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Cardiovasc. Pharmacol. 10 [Suppl4], S107-S109.
10
CHAPTER 1. Agonist-specific and non-specific in vitro desensi-
tization of human lymphocyte ß2-adrenoceptors
X-L Wang. LJH van Tits and NM Deiqhton
Biochemisches Forschungslabor, Medizinische Klinik und
Poliklinik, Abteilung für Nieren- und Hochdruckkrankheiten,
Universität Essen, D-4300 Essen, Fed. Rep. Germany
Running Title: Lymphocyte ß2-Adrenoceptor Desensitization
Key-Words: - ß-Adrenoceptors -
- Human Lymphocytes -
- Phorbol Esters -
- Homologous Desensitization -
- Heterologous Desensitization -
ABSTRACT — To study agonist-induced desensitization of human
ß-adrenoceptors we determined in vitro the effects of
catecholamines, prostaglandin E1 (PCE^ , and the phorbol ester
12-0-tetradecanoyl-phorbol-13-acetate (TPA) on lymphocyte
ß-adrenoceptor function. Incubation of lymphocytes in autologous
plasma at 37DC with catecholamines resulted in a time- and
concentration-dependent decrease in ß2-adrenoceptor density and
-responsiveness (cyclic AMP response to 10 μΜ isoprenaline); the
order of potency was isoprenaline > adrenaline > noradrenaline.
This catecholamine-induced decrease in ß2-adrenoceptor function
was completely abolished by the ß2-adrenoceptor antagonist ICI
118,551 (50 nM) , but not affected by the ß-adrenoceptor antagonist
bisoprolol (500 nM) . Incubation of lymphocytes with 10 μΜ PGEj
did not affect ß2-adrenoceptor function, but significantly
attenuated cyclic AMP responses to 10 μΜ PGEj. Incubation of
lymphocytes with 10 μΜ TPA had no effect on ß2-adrenoceptor
density, but markedly diminished cyclic AMP responses to 10 μΜ
isoprenaline and PGEj ; this effect was completely suppressed by
the protein kinase inhibitor H-7 (10 μΜ). In contrast, cyclic
AMP response to 10 μΜ forskolin was enhanced following TPA
incubation. It is concluded that human lymphocyte ß2-adrenoceptors
11
can undergo homologous (i.e. agonist-specific) and heterologous
desensitization. TPA-induced heterologous desensitization seems
to involve activation of protein kinase C.
INTRODUCTION
Desensitization, i.e. a diminished cellular responsiveness
following long-term exposure to agonists, seems to be a general
mechanism of cellular adaptation to agonist stimulation with time
(1-3). Two major types of desensitization have been described:
"homologous" desensitization results in an attenuated cellular
response only to stimulation of the desensitizing hormone and
seems to be cyclic AMP independent. In contrast "heterologous"
desensitization results in a diminished cellular response also
to additional ligands, and seems to be cyclic AMP dependent. For
the ß-adrenoceptor/adenylate cyclase system it has been shown in
various animal model systems, that after long time exposure to
ß-adrenoceptor agonists the ß-adrenoceptor responsiveness is
attenuated. The mechanism underlying this homologous desensi
tization has been intensively investigated in the last few years,
and the following concept has emerged, at least for high agonist
concentration (for references see 4-6): agonist activation of
the ß-adrenoceptor seems to induce translocation of the (cyto-
solic) enzyme ß-adrenoceptor protein kinase (ßARK) from the
cytosol to the plasma membrane to phosphorylate the receptor and
hence to uncouple it from the stimulatory guanine-nucleotide-
binding protein (Gg) . Accordingly, subsequent stimulation of the
receptor cannot result in activation of adenylate cyclase. This
"uncoupling,,-process is followed by a sequestration of the
receptor within the cell in a still unknown compartment resulting
in a decrease in the number of cell surface receptors.
However, it has recently been shown in turkey (7,8) and duck
erythrocytes (9) that in addition to ß-adrenoceptor agonists
phorbol esters - agents known to be potent activators of protein
kinase С (10) - can induce ß-adrenoceptor desensitization. This
heterologous desensitization seems to involve phosphorylation of
the ß-adrenoceptor by either the cyclic AMP dependent protein
12
kinase A or by protein kinase С (4-6) . Relatively little is known
about homologous and heterologous desensitization of human
ß-adrenoceptors. Circulating lymphocytes, containing a homo
geneous population of ß2-adrenoceptors excitatorily coupled to
the adenylate cyclase, are a frequently used model to study
ß-adrenoceptor changes in man (for recent reviews see 3,11,12).
The aim of this study was, therefore, to investigate whether
human lymphocyte ß2-adrenoceptors may underlie similar regulatory
mechanisms as have been demonstrated in the various mammalian
and avian model systems. For this purpose we determined in vitro
the effects of catecholamines, prostaglandin E-^ (PGEj) and the
phorbol ester 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on
lymphocyte ß2-adrenoceptor density (assessed by (-)-[ 125I]-io-
docyanopindolol (ICYP) binding) and on the lymphocyte cyclic AMP
response to 10 μΜ isoprenaline stimulation.
METHODS
For preparation of lymphocytes 100 ml EDTA-blood was taken from
healthy volunteers (aged 24-33 years) after 30 min of rest in
the supine position. The EDTA-blood was centrifuged at 2 50 g for
15 min at room temperature and plasma was carefully removed. The
remaining blood was diluted with an equal volume of phospha
te-buffered saline (PBS) and lymphocytes were isolated by the
method of Böyum (13), three times washed with PBS and finally
resuspended in autologous plasma to yield a concentration of
approximately 5xl06 cells per ml.
In vitro desensitization of lymphocyte ßn-adrenoceptors: Two
ml of the lymphocyte-suspension were incubated with the
catecholamines isoprenaline, adrenaline and noradrenaline in the
presence and absence of 50 nM ICI 118,551 or 500 nM bisoprolol,
or with PGE^ and TPA for the indicated times at 370C. The incubation
was stopped by rapid dilution of the samples with 10 ml of ice-cold
PBS and the whole reaction mixture was centrifuged at 400 g for
10 min. The resulting pellets were washed three times with 10 ml
ice-cold PBS and the lymphocytes were finally resuspended in
incubation buffer (12 mM Tris-HCl, 154 mM NaCl buffer pH 7.2,
13
that contained 30 μΜ phentolamine and 0.55 mM ascorbic acid; for
ICYP-binding) or in PBS containing 0.2% BSA and 100 μΜ theophylline
(for cyclic AMP determination). Lymphocyte ß2-adrenoceptor
density was assessed in intact cells by ICYP-binding at 6-8
concentrations ranging from 10-150 pM at 370C for one hour;
non-specific binding was measured in the presence of 1 μΜ of
(±)-CGP 12177. Lymphocyte cyclic AMP content was measured by the
protein binding assay of Gilman (14) as modified by Schwabe and
Ebert (15) . Details of the method have been described elsewhere
(16). In some experiments ß2-adrenoceptor density was determined
in lymphocyte membranes. For this purpose, after the final wash,
lymphocytes were lyzed in ice-cold hypotonic buffer (5 mM Tris-HCl,
5 mM EDTA buffer pH 7.4), homogenized at 40C with a glass teflon
homogenizer (Braun, Melsungen, F.R.G.), centrifugea at 50000 g
for 15 min and resuspended in incubation buffer (10 mM Tris-HCl,
154 mM NaCl buffer pH 7.4 containing 0.55 mM ascorbic acid).
Statistical evaluations: All data given in text and the figures
are means ± SEM of N experiments. The maximal number of binding
sites (Bmax) and the equilibrium dissociation constant (KD) for
ICYP were calculated either from plots according to Scatchard
(17) or by the iterative curve-fitting program LIGAND (18). Both
methods yielded identical results. Students t-test was used to
evaluate statistical significances; a P-value smaller than 0.05
was considered to be significant.
Drugs: (-)-Adrenaline bitartrate, (-)-Noradrenaline bitartrate,
(-)-Isoprenaline sulphate (IPN), Prostaglandin E1, TPA
(12-0-tetradecanoyl-phorbol-13-acetate) and Forskolin were
purchased from Sigma (München, F.R.G.), H-7
(1-(5-isoquinoline-sulfonyl)-2-methyl-piperazine) from Calbio-
chem (Frankfurt, F.R.G.). Bisoprolol hemifumarate (EMD 33,512;
bis-(1-(4-((2-isopropoxyethoxy)-methyl)-phenoxy)-3-isopropylam
ino-2-propanol) was kindly donated by Merck (Darmstadt, F.R.G.),
ICI 118,551 hydrochloride (erythro-(±)-1-(7-methylindan-4-ylox
y)-3-isopropylaminobutan-2-ol) by ICI-Pharma (Plankstadt,
F.R.G.) and (±)-CGP 12177 hydrochloride (4-(3-tertiarybutylami
14
no-2-hydroxy-propoxy)-benzimidazole-2-on) by CIBA-GEIGY (Basel,
Switzerland). (-)-[125I]-Iodocyanopindolol (specific activity
2200 Ci/mmol) and [3H]-cyclic AMP (specific activity 34.5 Ci/mmol)
were obtained from New England Nuclear (Dreieich, F.R.G.). All
other drugs were of the highest purity grade commercially
available.
RESULTS
B2-Adrenoceptor Desensitization Induced by Isoprenaline. Figure
1 shows the time course of the effects of 10 μΜ isoprenaline on
lymphocyte fl2-adrenoceptor density and lymphocyte cyclic AMP
response to 10 μΜ isoprenaline. Isoprenaline induced a rapid
decline in the lymphocyte cyclic AMP response amounting about
70% and 90% after 15 and 60 min respectively (Fig. 1A). On the
other hand, ß2-adrenoceptor density in the intact lymphocytes
declined much more slowly: it decreased by about 15% and 35%-40%
after 30 min and 2 hours, respectively (Fig. 1A). However, when
incubation was carried out for 24 hours ß2-adrenoceptor density
decreased to a similar extent as lymphocyte cyclic AMP response
(i.e. 85% reduction; data not shown). On the other hand, when
ß2-adrenoceptor density was assessed in lymphocyte membranes after
the isoprenaline incubation, a decrease of 35%, 50% and 70% was
obtained at 30, 60 and 120 min, respectively (Fig. IB) . The
KD-values of ICYP, however, were not affected by the isoprenaline
incubation; they amounted to 10-16 pM both in intact cells and
membranes.
Effects of Catecholamines on Lymphocyte ßj-Adrenoceptor Density.
Incubation of lymphocytes with isoprenaline (1 nM to 100 μΜ) and
adrenaline (10 nM to 100 μΜ) for 60 min at 370C resulted in a
concentration-dependent decrease in ß2-adrenoceptor density (Fig.
2) . Isoprenaline (EC50 = 41 nM) was 10 times more potent than
adrenaline (EC50 = 411 nM) . On the other hand, noradrenaline did
not affect lymphocyte ß2-adrenoceptor density in concentrations
up to 1 μΜ. At higher concentrations a slight reduction in
lymphocyte ß2-adrenoceptor density was observed (Fig. 2) . The
highly selective ß2-adrenoceptor antagonist ICI 118,551 (50 nM)
15
.900-1
l/l — О)
іл О.700-
•Ό
ç m CL
'500-
A. Intact Cells (N=4)
^ ^"AdrenoMpl·«· Density
'a> "S 25-о fe. СП E \
•о с э о .•
20-
15-
| ю-·
5-
ΙΟμΗ ΙΡΝ-evoked
CAMP Increose
ι — ι — ι — 1 — 1 — 1 — 1 — 1 — I
B. Membranes (N=4)
r12
-8
-U
Lo
•о 3 о
го Ι Λ
1-1
> 2 "Π
о с *
η η>
ьч
У -г-
30 60 Τ — г -
90 τ — г
120 min
Fig. 1. Time course of the effects of 10 μΜ isoprenaline on ß2" a d r e n o c eP t o r
density in both intact lymphocytes (A) and membranes (B), and 10 μΜ isopre-naline-induced cyclic AMP increase in intact lymphocytes (A). Lymphocytes
were incubated with 10 μΜ isoprenaline at 37<,C in autologous plasma and at
certain time intervals lymphocyte ß2-adrenoceptor density (squares) and the 10 μΜ isoprenaline- (IPN) induced cyclic AMP increase (triangles) were
determined. Each point is a mean value of 4 separate experiments. The vertical
bars represent SEM.
completely suppressed the isoprenaline (10 μΜ) -induced decrease
in lymphocyte ßj-adrenoeeptor density and cyclic AMP response
(Fig. 3); but the highly selective ß1-adrenoceptor antagonist
bisoprolol (500 nM) did not affect isoprenaline-induced effects
on lymphocyte ß2-adrenoceptor function (Fig. 4).
16
100-1
¡л 80-Ol о
О _^
о о
і£ "О
ΐχ 40-с
ч 10*9 10"В 10"7 10"6 10"5
[ß-Adrenergic Agonists] IO'4 M
Fig. 2. Influence of isoprenaline, adrenaline and noradrenaline on lymphocyte ß2-adrenoceptor density. Lymphocytes were incubated for 60 min at 370C with different concentrations of the catecholamines in autologous plasma and ßj-adrenoceptor density was determined. Each point is a mean value of 4 to 5 (given in parentheses) separate experiments. The vertical bars represent SEM.
17
1000η QJ
Ι Λ
g>500-
с m α. >-— Λ -
U"-
12-1 ι/)
^ а
ΙΛ
ω
I 4-α.
Λ_
(
JO μ
ΙοηΙτοΙ 10 μΜ ΙΡΝ 1
ι
Ι
4
Γ
4
ΟμΜ IPNU ІОпМІСІ
4
Μ IPN-evoked cAMP Increas
4
I * *
4
ι
4
e
Fig. 3. Effects of 50 nM ICI 118,551 (ICI) on 10 μΜ isoprenaline-evoked changes
in lymphocyte ß2 _ a^ r e n o c eP t o l : density (upper panel) and 10 μΜ isoprenaline-evoked lymphocyte cyclic AMP increase (lower panel) . Lymphocytes were incubated
for 60 min at 37eC in autologous plasma with 10 μΜ isoprenaline (IPN) in the
presence or absence of 50 nM ICI 118,551 and lymphocyte ß2-adrenoceptor density and 10 μΜ isoprenaline- (IPN) evoked cyclic AMP increase were determined. Given are mean values ± SEM; number of experiments are given at the bottom
of the columns. **) ρ < 0.01, *) ρ < 0.05 vs. the corresponding control (i.e.
lymphocytes incubated for 60 m m at 37°C in autologous plasma with PBS).
Effects of 10 μΜ Prostaglandin E1 (PGE
1) on Lymphocyte
fl2-Adrenoceptor Density. Incubation of lymphocytes for 60 min at
37"C with 10 μΜ PGEj^ neither affected lymphocyte ß2-adrenoceptor
density nor lymphocyte cyclic AMP response to 10 μΜ isoprenaline
18
ßo-Adrenoceptor Density
1000-1 Ol
LJ S . to cu
іТі ^500-
TD С
CD
CL
Control 10μΜ IPN 10μΜ IPN • 500 nM Biso
10μΜ IPN-evoked cAMP Increase
ΙΛ
~&
CD
\ ΙΛ Ol
"o E a.
12-1
8-
4-
ι * * .*»
Fig. 4. Effects of 500 nM bisoprolol (Biso) on 10 μΜ isoprenaline-evoked
changes in lymphocyte ß2-adrenoceptor density (upper panel) and 10 μΜ
isoprenaline-evoked lymphocyte cyclic AMP increase (lower panel). Lymphocytes
were incubated for 60 min at 370C in autologous plasma with 10 μΜ isoprenaline
(IPN) in the presence or absence of 500 nM bisoprolol and lymphocyte
ß2-adrenoceptor density and 10 μΜ isoprenaline- (IPN) evoked cyclic AMP
increase were determined. Given are mean values ± SEM; number of experiments
are given at the bottom of the columns. **) ρ < 0.01, *) ρ < 0.05 vs. the
corresponding control (i.e. lymphocytes incubated for 60 min at 37<,C in
autologous plasma with PBS).
(Fig. 5) . On the other hand, at this concentration PGE1 sig
nificantly decreased the lymphocyte cyclic AMP response to 10 μΜ
PGEi (Fig. 6).
19
1000-1
ш
¿Л
^500·
с CD
CL >-
f32~Adrenoceptor Density
Control 10gMPGE1
10 uM IPN-evoked cAMP Increase
"δ •о
о
о E Q.
12-1
8-
4-
Fig . 5. Effects of PGEj on lymphocyte n 2 ~ a d r e n o c e P t o r dens i ty (upper panel) and 10 μΜ isoprenal ine-evoked lymphocyte c y c l i c AMP increase (lower p a n e l ) . Lymphocytes were incubated for 60 min at 37°C with 10 μΜ PGEi in autologous plasma and lymphocyte ß2-adrenoceptor dens i ty and 10 μΜ i soprena l ine (IPN) -evoked c y c l i c AMP increase were determined. Given are mean values ± SEM; number of experiments are given a t the bottom of t h e columns.
E f f e c t s of the Fhorbol Ester TPA on Lymphocyte ß2-Adrenoceptor
Funct ion. I n c u b a t i o n of lymphocytes w i th 10 μΜ TPA f o r 30 min a t
37"С had no e f f e c t on lymphocyte ß2 -ad renocep to r d e n s i t y ( c o n t r o l :
996 ± 102 ICYP b i n d i n g s i t e s pe r c e l l , N=10; a f t e r TPA: 1072 ±
144 ICYP b i n d i n g s i t e s pe r c e l l , N=10); however, t h e b a s a l
lymphocyte c y c l i c AMP c o n t e n t ( c o n t r o l : 3.2 ± 0.4 pmol pe r 106
c e l l s , N=10; a f t e r TPA: 8.9 ± 2.2 pmol pe r 106 c e l l s , N=10, ρ <
2 0
Ίν
о
< (-1 ю Ol о E CL
2 5 -
20-
15-
10-
5-
0 -
10μΜ PGE-i-evoked Lymphocyte
cAMP Increase
Control 10 μΜ PGEÌ
* *
Fig. 6. Effect of 10 μΜ PGEi o n Ю M*1 PGEj-evoked increase in lymphocyte cyc l ic AMP c o n t e n t . Lymphocytes were incubated for 60 min a t 37°C with 10 μΜ PGEi i n autologous plasma and t h e 10 μΜ PGE^-evoked c y c l i c AMP i n c r e a s e was determined. Given a re mean values ± SEM; number of experiments a re given a t the bottom of t h e columns. **) ρ < 0.01 vs . c o n t r o l ( i . e . lymphocytes incubated in autologous plasma for 60 min a t 370C with PBS).
0.05) a s w e l l a s t h e lymphocyte c y c l i c AMP r e s p o n s e t o 10 μΜ
f o r s k o l i n ( F i g . 7) was s i g n i f i c a n t l y i n c r e a s e d . On t h e o t h e r
hand, t h e lymphocyte c y c l i c AMP r e s p o n s e t o 10 μΜ i s o p r e n a l i n e
o r 10 μΜ PGEj was r e d u c e d , a l t h o u g h t h i s d i d n o t r e a c h s t a t i s t i c a l
s i g n i f i c a n c e ( F i g . 7 ) . P r o l o n g i n g t h e i n c u b a t i o n of lymphocytes
w i t h TPA up t o 2 h o u r s r e s u l t e d i n a f u r t h e r d e c r e a s e i n 10 μΜ
i s o p r e n a l i n e - and 10 μΜ PGEj-induced c y c l i c AMP a c c u m u l a t i o n ,
w h i l e 10 μΜ f o r s k o l i n - i n d u c e d a c c u m u l a t i o n of c y c l i c AMP remained
a t t h e s t i m u l a t e d l e v e l ( F i g . 7) ; lymphocyte ß 2 - a d r e n o c e p t o r
d e n s i t y , however, was s t i l l unchanged ( a f t e r 2 hou r s TPA: 889 ±
122 ICYP b i n d i n g s i t e s pe r c e l l , N=5). The e f f e c t s of 2 hour s
i n c u b a t i o n w i t h TPA on lymphocyte c y c l i c AMP r e s p o n s e t o 10 μΜ
i s o p r e n a l i n e o r 10 μΜ PGEÍ were comple te ly a b o l i s h e d by t h e p r o t e i n
k i n a s e С i n h i b i t o r H-7 (10 μΜ, F i g . 8) .
21
Α. I PN
10η
u, 8 -<u
l_» v O
О /· ^; 6-α. Σ: <
Ol
о E о. _
2-
л _
Control ι
ΤΡΑ
30min
9
120m
ι
9
Г
5
Control ΤΡΑ
20π
η
16
1 2 -
8 -
4-
0-
30min 2 0 ~ Ι
120min
16
12-
8 -
CForskotin Control ΤΡΑ
30min 120min
10 10
Fig. 7. Effect of ΤΡΑ on lymphocyte cyc l ic AMP response t o 10 μΜ i s o p r e n a l i n e , PGEj and f o r s k o l i n . Lymphocytes were incubated in autologous plasma for 30 and 120 mm, r e s p e c t i v e l y , a t 370C with 10 μΜ ΤΡΑ and t h e 10 μΜ i s o p r e n a l i n e -(IPN), PGEi-, or forskolin-evoked increase in lymphocyte c y c l i c AMP content was determined. Given are mean values ± SEM; number of experiments are given a t t h e bottom of t h e columns. **) ρ < 0 .01, *) ρ < 0.05 vs. control ( i . e . lymphocytes incubated in autologous plasma for 30 mm at 370C with PBS).
DISCUSSION
I n t h e p r e s e n t s t u d y , c a t e c h o l a m i n e s caused d e s e n s i t i z a t i o n of
lymphocyte ß 2 - a d r e n o c e p t o r s wi th an o r d e r of po tency t y p i c a l fo r
ß 2 - a d r e n o c e p t o r s ( 1 9 , 2 0 ) : i s o p r e n a l i n e > a d r e n a l i n e > n o r a d r e
n a l i n e . The h i g h l y s e l e c t i v e ß 2 - a d r e n o c e p t o r a n t a g o n i s t ICI
118,551 (21) comple te ly a b o l i s h e d i s o p r e n a l i n e - i n d u c e d
d e s e n s i t i z a t i o n of lymphocyte ß2 -ad renocep to r f u n c t i o n , wh i l e t h e
h i g h l y s e l e c t i v e ß 1 - a d r e n o c e p t o r a n t a g o n i s t b i s o p r o l o l (22) a t
a c o n c e n t r a t i o n t h a t occup ie s about 95% of ß j - a d r e n o c e p t o r s and
20% of ß 2 - a d r e n o c e p t o r s (23) , had no e f f e c t . These r e s u l t s s t r o n g l y
s u p p o r t t h e view t h a t t h e c a t e c h o l a m i n e - i n d u c e d d e s e n s i t i z a t i o n
of lymphocyte ß - a d r e n o c e p t o r s i s a s e l e c t i v e ß2 -ad renocep to r
dependent p r o c e s s .
22
10-
^ 8-Α ι
(_J vO
СЭ
S 6-Σ: <
Й 4-e CL
2-
л_
ΑΙΡΝ В. PGEÏ
Control ΙΟμΜΤΡΑ TFtt Control ΒμΜΤΡΑ TRI\
+ЮиМН-7 •«ui I . 30η
5
J
Г*
5
n.s
Π 24-
18-
12-
6-
5 л.
ι— П!
г·
5 5 5
Fig. θ. Effect of H-7 on TPA-effects on lymphocyte cyclic AMP response to 10
μΜ isoprenaline or PGE}. Lymphocytes were incubated in autologous plasma for
120 min at 370C with 10 μΜ TPA in the presence or absence of 10 μΜ H-7 and
the 10 μΜ isoprenaline- (IPN) or PGE^-evoked increase in lymphocyte cyclic
AMP content was determined. Given are mean values ± SEM; number of experiments
are given at the bottom of the columns. **) ρ < 0.01, n.s. = not significantly
different vs. control (i.e. lymphocytes incubated in autologous plasma for
120 min at 37°C with PBS).
Whereas isoprenaline caused a rapid decrease in the lymphocyte
cyclic AMP response to isoprenaline stimulation, the density of
ß2-adrenoceptors declined more slowly. On the other hand, PGEj
(a potent activator of lymphocyte adenylate cyclase, 24) did not
affect lymphocyte ß2-adrenoceptor density and cyclic AMP response
to isoprenaline, but significantly decreased lymphocyte cyclic
AMP responses to PGEj stimulation. These results demonstrate that
lymphocyte ß2-adrenoceptors undergo ß-adrenoceptor agonist
induced homologous desensitization similar to those demonstrated
for ß-adrenoceptors in various animal model systems (see
introduction) .
23
It is, therefore, tempting to speculate that lymphocyte
ß2-adrenoceptors after activation by μΜ concentrations of iso
prenaline may be phosphorylated by the enzyme ßARK resulting in
an uncoupling of the receptor from the Gs-protein/adenylate
cyclase complex later followed by sequestration of the receptors
within the cell (4,5). This assumption is strongly supported by
the fact that after 15 min of incubation with isoprenaline, the
lymphocyte cyclic AMP response to isoprenaline was decreased by
about 70%, while ß2-adrenoceptor density was only slightly
reduced. In this respect, it is worthwhile to note that lymphocyte
ß2-adrenoceptor density decreased more rapidly following iso
prenaline incubation, when determined in membranes than in intact
cells. This may be due to the fact, that in the present study
intact lymphocytes were incubated with the ß-adrenoceptor
radioligand ICYP for a rather long period (60 min); during this
period it is possible that part of the desensitized ß2-adreno-
ceptors may be resensitized (25), thus masking a more pronounced
decrease in receptor number. Furthermore, the present results
clearly indicate, that homologous desensitization of ß-adreno-
ceptors in human lymphocytes is also cyclic AMP independent (4-6)
since after incubation with PGE1 (which produces in lymphocytes
marked increases in cyclic AMP content) at a concentration
sufficient to desensitize PGE1-mediated effects (cf. Fig. 6)
neither lymphocyte ß2-adrenoceptor density nor lymphocyte cyclic
AMP response to isoprenaline stimulation were affected.
It has recently been reported that the tumor promoting phorbol
ester TPA, a potent protein kinase С activator (10), can affect
the ß-adrenoceptor/adenylate cyclase system. The effects of TPA
on ß-adrenoceptor function, however, depend on the cell type
studied: it increased ß-adrenergic activation of adenylate cyclase
in S49 lymphoma cells (26,27) and frog erythrocytes (28) but
diminished responses of the adenylate cyclase to isoprenaline in
rat reticulocytes (29), C6 glioma cells (30), and in turkey (7,8)
and duck erythrocytes (9). In the present study, incubation of
human lymphocytes with TPA resulted in an increase in basal cyclic
AMP content and an enhanced cyclic AMP response to forskolin (cf.
24
Fig. 7) , which directly activates the catalytic unit of the
adenylate cyclase (31). A similar augmentation of the fors-
kolin-induced activation of adenylate cyclase following incu
bation with TPA has recently been observed in several intact
cells and cell-free preparations (26,27,32-34). The effect was
ascribed to a protein kinase C-induced phosphorylation of the
inhibitory guanine-nucleotide-binding regulatory component of
adenylate cyclase (G^), which results in the suppression of the
tonic inhibition of adenylate cyclase activity (35,36).
Incubation of lymphocytes with TPA did not affect lymphocyte
ß2-adrenoceptor density, but led to a marked reduction in lym
phocyte cyclic AMP response to ß-adrenergic (isoprenaline) as
well as PGEj^-stimulation. These effects seemed to be mediated by
protein kinase С activation since they could be completely
prevented by simultaneous incubation of the lymphocytes with the
protein kinase С inhibitor H-7 (37) . Similar results have recently
been described by Meurs et al. (38) who observed in human
mononuclear leucocytes that TPA did not affect ß-adrenoceptor
density, but caused a concentration-dependent desensitization of
isoprenaline-, histamine- and PGEj^-stimulated adenylate cyclase
activity. The mechanism of heterologous desensitization of the
human lymphocyte ß-adrenoceptor/adenylate cyclase system induced
by phorbol esters is not known at present. However, it might be
due to an impairment of Gg-protein function and/or the ability
of the ß-adrenoceptor to couple to Gs. Further studies are
necessary to elucidate the precise mechanism of phorbol
ester-induced heterologous desensitization of the human lym
phocyte ß-adrenoceptor/adenylate cyclase system.
In summary: the present results clearly demonstrate that
ß2-adrenoceptors in circulating lymphocytes undergo homologous
and heterologous desensitization in a manner which is similar to
that observed in various mammalian and avian model systems. Since
the properties of lymphocyte ß2-adrenoceptors resemble those of
ß2-adrenoceptors in other human tissues (eg. human heart and human
saphenous vein; see 11) , and since it has recently been shown
25
that lymphocyte ß2-adrenoceptor changes can mirror precisely
ßj-adrenoceptor changes in solid human tissues like heart (39),
myometrium (40) or lung (41) , it is concluded that lymphocyte
ß2-adrenoceptors are an useful model for studying the regulation
of human ß2-adrenoceptors in vitro.
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Protein kinase С phosphorylates the inhibitory guanine
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37. Hidaka H, Inagaki M, Kawamoto S and Sasaki Y. Isoquinoline
sulfonamides, novel and potent inhibitors of cyclic nucleotide
dependent protein kinase and protein kinase C. Biochemistry 1984;
23: 5036-5041.
38. Meurs H, Kauffmann HK, Timmermans A, Van Amsterdam FTM, Koeter
GH and Vries KD. Phorbol 12-myristate 13-acetate induces
beta-adrenergic receptor uncoupling and non-specific desensi-
tization of adenylate cyclase in human mononuclear leucocytes.
29
Biochemical Pharmacology 1986; 35: 4217-4222.
39. Michel MC, Pingsmann A, Beckeringh JJ, Zerkowski Η-R, Doetsch
N and Brodde 0-E. Selective regulation of ß1- and ßj-adrenoceptors
in the human heart by chronic ß-adrenoceptor antagonist treatment.
British Journal of Pharmacology 1988; 94: 685-692.
40. Michel MC, Pingsmann A, Nohlen M, Siekmann U and Brodde O-E.
Decreased myometrial ß-adrenoceptors in women receiving
ßj-adrenergic tocolytic therapy: correlation with lymphocyte
ß-adrenoceptors. Clinical and Pharmacological Therapeutics 1989;
45: 1-8.
41. Liggett SB, Marker JC, Shah SD, Roper CL and Cryer PE. Direct
relationship between mononuclear leukocyte and lung ß-adrenergic
receptors and apparent reciprocal regulation of extravascular,
but not intravascular, a- and ß-adrenergic receptors by the
sympathochromaffin system in humans. Journal of Clinical
Investigation 1988; 82: 48-56.
30
CHAPTER 2. Catecholamines increase lymphocyte ßj-adrenergic
receptors via a ßj-adrenergic, spleen-dependent
process
L.J.H. van Tits, M.C. Michel, H. Grosse-Wilde, M. Happel, F.-W.
Eigler, A. Soliman, and O.-E. Brodde.
Department of Pharmacology M-036, University of California at
San Diego, La Jolla, California 92093; Biochemical Research
Laboratory, Medizinische Klinik und Poliklinik, Institut für
Immungenetik, Zentrum für Chirurgie, Chirurgische Klinik und
Poliklinik, University of Essen, D-4300 Essen, Federal Republic
of Germany
ß2-adrenergic receptors on lymphocyte subsets; spleen and lym
phocyte subsets; lymphocyte immune function; mitogens
ABSTRACT
We investigated the mechanisms underlying the increase in
mononuclear leukocyte (MNL) ß2-adrenergic receptor (AR) number
and responsiveness after acute infusion of catecholamines.
Infusion of isoproterenol and epinephrine, but not of norepi
nephrine, acutely increased MNL ß2-adrenoceptor density, and this
was blocked by the ß2-selective antagonist ICI 118,551 but not
by the ßa-selective antagonist bisoprolol, suggesting a ß2-AR-
mediated effect. Infusion of isoproterenol but not of norepi
nephrine also induced a lymphocytosis, with an increase in the
number of circulating suppressor/cytolytic Τ (Ts/
C)- and natural
killer (NK)-cells but a decrease in helper Τ (Th)-cells, leading
to a decreased Th-/T
s/
c-cell ratio. ß-AR density was higher in
Ts/c-cells than in Th-cells. After isoproterenol infusion, ß-AR
density was elevated in all lymphocyte subsets but not in monocytes
or platelets, suggesting a lymphocyte-specific phenomenon.
Infusion of isoproterenol in splenectomized patients did not
alter lymphocyte subset composition and only slightly increased
ß2-AR density. In healthy subjects lymphocyte proliferation in
response to various mitogens was attenuated after infusion of
isoproterenol but not of norepinephrine; this effect was abolished
31
in splenectomized patients. We conclude that the elevated MNL
ß-AR density after acute exposure to ß-adrenergic agonists is
caused by a release of lymphocyte subsets from the spleen into
the circulation and/or by an exchange of lymphocyte subsets
between the spleen and the circulation, whereby freshly released
splenic lymphocytes appear to carry more ß-AR than those found
in the circulation. This appears to impair immune responsiveness
in a dual manner, by decreasing the Th-/Tg//c-cell ratio and by
rendering lymphocytes more sensitive to the antiproliferative
effects of catecholamines via a higher ß-AR density.
INTRODUCTION
GROWING EVIDENCE SUGGESTS that the immune system is at least
partly under the control of the sympathetic nervous system. Immune
responsiveness is altered in various animal models of stress (31,
32, 39, 43). In humans, conditions of chronically heightened
sympathetic activity, like congestive heart failure, can also be
associated with an altered immune status (3, 25, 35). However,
little is known about the mechanisms by which the sympathetic
nervous system can modulate immune function.
The sympathetic nervous system and the immune system are
anatomically linked by a dense innervation of the spleen and
other immunological tissues. In these tissues, lymphocytes and
sympathetic nerve endings form contacts at a distance that is
even shorter than that in a synapse (for review see Ref. 27).
The presence of surface receptors for the sympathetic neuro
transmitters makes lymphocytes susceptible to sympathetic
stimulation. These receptors are of the ß2-adrenergic subtype
and couple to stimulation of adenylate cyclase (for review see
Ref. 13). In vitro, stimulation of ß2-adrenergic receptors (AR)
(by isoproterenol) and other adenylate cyclase-linked receptors
can inhibit lymphocyte proliferation, mitogen-induced secretion
of interleukin 2 (IL-2), and expression of IL-2 receptors (6,
26, 33) . ß-AR agonists might also have an antiproliferative effect
in vivo, as isoproterenol treatment reduces the number of thymic
32
lymphocytes in mice (24) . Whether the same holds true for plasma
catecholamines in physiological concentrations, however, remains
unclear at present.
The density of lymphocyte ß2-AR is not static but is dynamically
regulated by a wide variety of neurotransmitters and hormones,
physiological and pathophysiological conditions (10, 45) , making
a comparison with other solid human tissues questionable. As in
most other tissues (30) , long-term administration of ß-AR agonists
(e.g., procaterol, terbutaline, hexoprenaline) decreases the
number and responsiveness of ß-AR on circulating human lymphocytes
(4, 11, 40, 42). This ß-AR downregulation might protect the
lymphocyte against antiproliferative effects of a prolonged
exposure to ß-AR agonists. Acute stimulation of the activity of
the sympathetic nervous system by dynamic exercise or mental
arithmetic stress, however, increases the ß-AR density on
circulating human lymphocytes (12, 16, 17, 22, 29). This increase
can also be demonstrated by infusion of isoproterenol (15,48)
and can be blocked by propranolol (14), suggesting that it is
mediated by a ß-AR. Such a receptor increase might render
circulating lymphocytes more sensitive to the antiproliferative
effects of ß-AR agonists.
The mechanism underlying the increase in lymphocyte ß-AR after
dynamic exercise or catecholamine infusion in vivo is not well
understood. It could be because of an increase in ß-AR in some
or all of the circulating mononuclear leukocytes (MNL), or it
could be caused by differences between the MNL obtained before
and after catecholamine exposure. The rapid time course (within
15 min) makes de novo synthesis of ß-AR very unlikely. It has
been postulated that receptors might be uncovered that may have
been blocked before (47) . A variation of this hypothesis is the
idea that lymphocyte ß-AR can exist in several cellular com
partments (21, 44) , and acute sympathetic stimulation might shift
them from sequestered compartments to the cell surface. A third
possibility is based on the finding that subsets of circulating
lymphocytes differ in their ß-AR density (34, 36) and that acute
33
sympathetic stimulation might selectively alter the homing
properties of lymphocyte subsets releasing them into or removing
them from the circulation, thus mimicking ß-AR regulation.
The present study was designed to investigate the mechanisms
responsible for the increase of lymphocyte ß-AR after acute ß-AR
stimulation and the possible immunological consequences of such
alterations. Therefore, we infused isoproterenol, epinephrine,
and norepinephrine into healthy volunteers and splenectomized
patients. Before and after infusion, the MNL ß2-AR density and
responsiveness and the subset composition of circulating MNL were
determined. Additionally, we measured lymphocyte in vitro pro
liferative response to various mitogens before and after cate
cholamine infusion.
METHODS
Volunteers and infusion scheme. Infusion tests were performed
in 62 healthy volunteers (49 males and 13 females, age 26.3 ±
0.7 (22-34) yr) after having given informed written consent. All
volunteers were drug free for at least 3 wk before study, and
had undergone physical and electrocardiogram examination to
exclude signs of cardiovascular or pulmonary diseases. The
isoproterenol infusions were repeated in a subgroup of 10 male
healthy subjects [age 25.9 ±0.5 (24-28) yr], who had received
50 mg of the ß2-selective antagonist ICI 118,551 (5) or 30 mg of
the ß1-selective antagonist bisoprolol (9) orally 4 or 2.5 h
before the test, respectively. This timing and dosing of the two
selective ß-adrenergic antagonists were chosen because they cause
plasma levels corresponding to an occupancy of ~ 75-85% of lii
(bisoprolol) and ß2-AR (ICI 118,551) , but < 10% of ßj- (bisoprolol)
and ßj-AR (ICI 118,551), respectively (15,50).
Additional infusion tests were performed in five male patients
(age 30 ± 2 yr) who had undergone splenectomy because of Hodgkin's
disease (4 patients) or trauma (1 patient). The time interval
between splenectomy and isoproterenol infusion was 8-15 days for
the Hodgkin's patients and 15 days for the non-Hodgkin's patient,
respectively. Before, during, and after the surgery, patients
34
зосн • - • Control A - i Bisoprolot (BOmgpo
2 5h before Infusioni · - · IC1118551 (50mg po
í.h before Infusion)
~i— Ì5
— ι — 175
— ι 70 0 Ì5 7 175 35
Isoproterenol lng/kg/min for 5mm each)
• - a Control І - Д Bisoprolol (30mgpo
2 5h before Infusion) · - · IC1118551 (50mg po
4h before Infusion)
FIG. 1. A: effects of isoproterenol
infusion on lymphocyte ІІ2-adrenergic receptor (AR) density in 10 male
healthy volunteers in presence or
absence of bisoprolol (30 mg orally
2.5 h before infusion) or ICI 118,551
(50 mg orally 4 h before infusion).
Isoproterenol was infused in doses of
3.5, 7, 17.5, 35 and 70 ng kg- 1 m m
-!
for 5 min each; immediately before
infusion and after 10, 15, 20 and 25
m m of infusion hepannized blood was
withdrawn for lymphocyte ß2-AR determination. Ordinate: lymphocyte ß2-AR density, determined by Scatchard analysis (46) of i-flSSjj. lodocyanopindolol binding in intact cells, in percent of preinfusion level (= 100%). Abscissa: dose of isoproterenol in ng kg-l m m - 1 for 5 min each. B: effects of isoproterenol infusion on 10 μΜ isoproterenol
(ISO)-induced in vitro cAMP increase
in 10 male healthy volunteers in
presence of bisoprolol (30 mg orally
2.5 h before infusion) or ICI 118,551
(50 mg orally 4 h before infusion).
For details see A. Ordinate: 10 μΜ
isoproterenol-induced in vitro
increase in cAMP content in intact
cells in percent of preinfusion level
(= 100%). Abscissa: dose of
isoproterenol in ng kg- 1 m m
- 1 for 5
min each. Values are means ± SE. ** Ρ < 0.01, * Ρ < 0.05 vs. control.
35 175 35 70 Isoproterenol (ng/Kg/mm for 5 mm each)
did not receive ß-adrenergic agonists or antagonists. However,
the non-Hodgkin's patient received immediately after splenectomy
a small dose of dopamine (3 дд kg-l-min"! for 2 days) to maintain
renal function.
After an initial rest period of 1 h in the supine position,
isoproterenol (sequential doses of 3.5, 7, 17.5, 35 and 70
ng· kg-l-πύη"1 for 5 min each), epinephrine (50 ng kg-i-min"! for
35
90 min) or norepinephrine (50 ng kg-1 min
-1 for 90 min) were
infused. Blood pressure and heart rate were recorded automatically
by a Tonomed (Speidel & Keller, Jungingen, FRG) and an elec
trocardiogram. If not otherwise stated, immediately before and
after infusion, blood was withdrawn with 500 IU heparin/10 ml
blood.
= 1900
Э, isooi en Ç
I 1100 a. >-- 700
Epinephrine (50 ng/kg/mm)
• A Intact Cells (n=16i ^
Γ f f
.2 70-о CL
f 50· Ό С
È • CL ЗОН
e 10
с : : : : : : ι 1 1 1
θ Membranes (nrS)
f f f r ; : : : : ;
^ -f—
30 τ -
60
Norepinephrine fi0nq/kq/mm)
1500η i A Intact Cells (пгб) i
α.
1250-
1000-
Ε ä - ^ г
¿ 35-1
СП
ε •о с
s a >-
25-
15-
90min
- t - t - i В MembraneslOï^)
- ι 1 1 30 60 90mm
FIG. 2. Effects of epinephrine infusion
on lymphocyte ß2-adrenergic receptor (AR) density in 21 healthy volunteers. Ordinatesi Д, lymphocyte П2-АК den
sity, determined by Scatchard analysis
(46) of i-[125I]-iodocyanopindolol
(ICYP) binding in intact cells, in ICYP
binding sites/cell; B, lymphocyte
ß2-AR density, determined by Scatchard analysis (46) of ICYP binding in membranes, in fmol ICYP specifically bound/mg protein. Abscissas: time of infusion in min. Values are means ± SE; η = no. of experiments. Horizontal lines and broken lines, means ± SE of
preinfusion levels. Ρ < 0.01, * Ρ < 0.05 vs. corresponding preinfusion levels.
FIG. 3. Effects of norepinephrine
infusion on lymphocyte Л2""а с*
г е п е г9
1 С
receptor (AR) density in 10 healthy
volunteers. Ordinatesi Л, lymphocyte
ß2-AR density, determined by Scatchard analysis (46) of i-t^Sij-lodocyanopindolol (ICYP) binding in intact cells, in ICYP binding sites/cell; B, lymphocyte ß2-AR density, determined by Scatchard analysis (46) of ICYP binding in membranes, in fmol ICYP specifically bound/mg protein. Abscissas: time of infusion in min. Values are means ± SE; л = no. of experiments. Horizontal lines and
broken lines, means ± SE of preinfusion
levels.
36
Cell isolation. Heparinized blood was centrifuged for 15 min
at 300 g, and platelet-rich plasma was removed. In some experiments
platelets and platelet membranes were prepared from the plasma
as described previously (49) . MNL were prepared by Ficoll gradient
centrifugation of the pellet according to the method of Böyum
(7) with minor modifications (11). For monocyte isolation
RPMI-1640 medium was used to dilute the blood to four times the
original volume before the Ficoll centrifugation. For lymphocyte
subset isolation phosphate buffered saline (PBS) without Ca2+ and
Mg2+ was used. The recovery of leukocytes and lymphocyte subsets
as given below was quantified microscopically with a Neubauer
counting chamber.
Purified lymphocyte subsets were prepared from the final MNL
pellet by a panning technique by means of negative selection with
monoclonal antibodies. Briefly, MNL were suspended in 5% fetal
calf serum (FCSJ-PBS, and transferred into petri dishes that had
been coated with appropriate monoclonal antibodies. A mixture of
0KB2, Bl, and JotlO was used for isolation of pan-T-cells; of
Jot8, Jot4, JotlO, LeuMl, and 0KT16 for isolation of B-cells; of
0KB2, Jot8, JotlO, and Bl for isolation of helper Τ (Th)-cells;
and of OKB2, Jot4a, JotlO, and Bl for isolation of suppressor
/cytolytic Τ (Ts;c)-cells. The dishes were incubated at room
temperature for 1 h. Nonadherent cells were then carefully
collected, washed once, and transferred into another set of Petri
dishes for a repetition of the purification process. T-, Th- and
Ts/c-subsets isolated with this technique had a purity of >95%,
and B-cells of >85%, as determined by indirect immunofluorescence
(see below).
Monocytes were isolated from MNL by adherence to plastic.
Briefly, the MNL pellet was suspended in 20% FCS-RPMI 1640 medium
and transferred into plastic Petri dishes. The dishes were
incubated at 37 "С in a humidified atmosphere of 5% COj in air
for 1 h. The nonadherent cells were then decanted and the dishes
were gently washed. Adherent cells were collected by vigorous
pipetting after incubation with 0.8% Xylocaine in 20% FCS-RPMI
37
TABLE 1. R e i a t i v e and absolute distribution of leukocytes in 10 healthy
subjects before and after isoproterenol infusion
Leukocytes
Monocytes
Lymphocytes
B-cells
T-cells
Th-cells
TB/
c-cells
NK-cells
Th-to-Tg/c cell ratio
Isoproterenol Infusion
Bef
Rel (%)
3.2±0.б
41.3+2.6
14.1±1.0
76.311.2
43.5±1.0
26.4±1.6
3.8±0.6
ore
Abs
(cells/μΐ)
5095±547
2055±210
285+31
1556±149
879191
520+46
72 + 10
1.7±0.1
After
Rel (%)
3.411.0
50.415.2
11.0+1.2
78.2+1.7
20.511.9
41.3+1.6
12.3+2.2
Abs
(cells/μΐ)
53901593
2490+137*
274131
19431105*
506146**
1024160**
301149**
0.5+0.1**
Values are means i SE of 10 experiments. I soproterenol was infused in doses of 3.5, 7, 17.5, 35 and 70 ng kg _ l m i n - l for 5 min each; immediately before infusion and a f t e r 25 min of infusion h e p a n n i z e d blood was withdrawn for determinat ion of d i s t r i b u t i o n of monocytes and lymphocyte subse t s . Rel %, r e l a t i v e %; abs, a b s o l u t e ; Th, helper T - c e l l ; Ts/C, suppressor/cyto ly t ic T - c e l l ; NK, n a t u r a l k i l l e r . ** Ρ < 0 .01, * Ρ < 0.05 vs. t h e corresponding pre infus ion l e v e l s .
1640 for 20 min a t 4 0 C. Cell suspensions i s o l a t e d with t h i s
technique had a p u r i t y of >95% as determined with presta ined
s l i d e s for d i f f e r e n t i a l blood counts (Boehringer, Mannheim, FRG) .
MNL and enriched lymphocyte subpopulations were analyzed by
i n d i r e c t immunofluorescence with the following monoclonal
a n t i b o d i e s : 0KT3, J o t 3 , and Leu5b for pan-T c e l l s ; Jot4a, Leu3,
and BMA040 for T h - c e l l s ; Jot8a, Leu2, and TÜ102 for T s / c - c e l l s ; TÜ39 and aDR for B-ce l l s ; JotlO and Leu7 for natura l k i l l e r (NK)-cells; Tiik-l for monocytes. Fluorescein isothiocyanate (FITC)-labeled goat anti-mouse immunoglobulin G (IgG) + IgM was used as the second antibody.
38
TABLE 2. Relative distribution oí monocytes and lymphocyte subsets in 5 healthy subjects before, during, and alter norepinephrine infusion
Monocytes
B-cells
T-cells
Th-cells
Tg/c-cells
NK-cells
Th-to-Tg/c cell ratio
Norepinephrine Infusion
Before
rel %
4.2±0.5
15.4±1.0
78.Oil.8
41.8±1.9
31.6±1.8
3.2±0.5
1.4±0.1
30 min
rel %
4.0±0.4
16.2+1.1
77.4±1.6
39.6±1.4
30.2±1.5
3.0±0.5
1.3±0.1
60 m m
rel %
5.2±1.3
16.211.5
77.8±1.4
40.4±1.2
31.6±2.2
3.6±0.2
1.3±0.1
90 min
rel %
5.0±1.3
18.4±2.5
76.811.7
40.4±1.2
30.0±1.4
3.810.4
1.410.1
Values are means i SE of 5 experiments. See Table 1 for abbreviations. Norepinephrine was infused at a dose of 50 ng kg-l min-^ for 90 min; immediately before infusion and after 30, 60, and 90 min of infusion, hepannized blood was withdrawn for determination of distribution of monocytes and lymphocyte subsets.
Determination of ß-AR density and responsiveness. Lymphocyte
and monocyte ßj-AR were identified in intact cells by radioligand
binding with [125I]iodocyanopindolol (ICYP) at 6-8 concentrations
of ICYP ranging from 10 to 150 pM (11). It should be noted that
our assay conditions detect very likely only cell surface ß2-AR,
as we perform the assay in the presence of 30 μΜ phentolamine
(that inhibits uptake of ICYP into the cells, for references see
Ref. 10) and define non-specific binding by 1 μΜ of the hydrophylic
ß-AR antagonist (±)-CGP 12177. However, a redistribution of
sequestered receptors to the cell surface during the incubation
period (1 h, 37 0C) cannot be excluded. Thus additional experiments
were performed in cell membrane preparations under similar
conditions. Platelet ß2-AR were labeled with [ 125I]iodopindolol
(IPIN) according to previously published techniques (49). MNL,
pan-T-, and B-cell ß2-AR were determined in the blood of 5 subjects,
and monocyte, Th- and Tg/c-cell ß-AR in the blood of another 5
subjects, because we were not able to isolate all six fractions
in sufficient quantities from 200 ml of blood.
39
The accumulation of adenosine S'.S'-cyclic monophosphate (cAMP)
in intact cells during a 10-min incubation in the absence and
presence of 10 μΜ isoproterenol was measured according to
previously published techniques (11) .
0-« ' • ' — - (H r- 1— 1 — гт г-; ь0 CD before ISO Ю8 IO7 W^ t ) 5 Ю 4 М E¡a öfter ISO I Isoproterenol]
FIG. 4. Effects of isoproterenol (ISO) infusion on lymphocyte ß2-adrenergic receptor (AR) density (A) and isoproterenol-induced in vitro activation of lymphocyte adenylate cyclase (B) in 5 male healthy volunteers. Isoproterenol was infused in doses of 3.5, 7, 17.5, 35 and 70 ng kg-l min-^ for 5 min each; immediately before infusion and after 25 m m of infusion hepannized blood was withdrawn for determination of lymphocyte ß2-AR density and adenylate cyclase activity. Ordinates: A, lymphocyte ß2-AR density, determined by Scatchard analysis (46) of J-[125j]-iodocyanopindolol (ICYP) binding in membranes, in fmol ІСУР specifically bound/mg protein; B, adenylate cyclase activity in lymphocyte membranes expressed either in pmol cAMP formed mg
protein-* min
-l or in percent of basal activity (=100%) before (ieft) and
after isoproterenol infusion (right). Abscissa in B: molar concentrations of
isoproterenol. Values are means ± SE; no. of experiments in parentheses. **
Ρ < 0.01, * Ρ < 0.05 vs. corresponding preinfusion levels.
Adenylate cyclase activity was measured in MNL membranes. The
MNL pellet was frozen in liquid nitrogen and stored at -80 0C
for maximally 8 days. No loss of adenylate cyclase activity was
observed during this time interval. At the day of the assay, the
pellet was suspended in 1 ml of buffer [10 mM tris(hydroxyme-
thyl)-aminomethane (Tris)-HCl, 2 mM МдСІ2, 0.1 mM ethylene
glycol-bisiß-aminoethyletherJ-N^N' ,N' (EGTA) , pH 7.4] and three
40
times freeze-thawed in liquid nitrogen. The resulting suspension
was diluted to a volume of 10 ml with buffer, centrifuged at
29,000 g for 10 min at 4 "С, and the pellet finally suspended
again in buffer. Membranes (2-6 дд of protein) were incubated at
37 0C for 10 min in total volume of 100 μΐ containing 25 mM
Tris-HCl buffer, 5 mM MgClj, 0.5 mM ATP, 5 mM creatine phosphate,
50 lU/ml creatine Phosphokinase, and 10 μΚ guanosine 5'-tris-
phosphate at pH 7.4, in the absence or presence of isoproterenol.
The reaction was stopped by immersing the incubation tubes in
boiling water for 5 min. After cooling, samples were centrifuged
at 12,000 g for 10 min and the cAMP concentration was determined
in aliquots of the supernatant with a radio-immunoassay kit, by
means of the nonacetylated procedure. Protein was determined by
the method of Bradford (8) with bovine serum albumin as a standard.
WOO-
=S 800-
£ 6 0 0 -
1 iOO-
Q.
δ 200-
A Intact Cells (n=5) SO-i
I to
so·
-о 20-
ó ЮН e
В Membranes (n=5)
Л
ΠΏ before Incubation ШЗ after Incubation (60 mm, 37βΟ
FIG. 5. Effects of in vitro incubation (60 min, 37 0C) of lymphocytes obtained
immediately before isoproterenol infusion, with autologous plasma, obtained
immediately after isoproterenol infusion (3.5, 7, 17.5, 35, and 70 ng -kg-! -min
-*
for 5 min each) on ß2-adrenergic receptor (AR) density. Ordinates: A, lymphocyte ß2-AR density, determined by Scatchard analysis (46) of i-[125j]_ iodocyanopindolol (ICYP) binding in intact cells, in ICYP binding sites/cell; B, lymphocyte ß2-AR density, determined by Scatchard analysis (46) of ICYP binding in membranes, in fmol ICYP specifically bound/mg protein. Values are means ± SE; л = no. of experiments.
Functional mononuclear cell tests. Monuclear cell function was
monitored by the ability of cells to proliferate (assayed by
[3H]thymidine incorporation) on mitogenic stimulation with
41
phytohemagglutinin (PHA; 0.45, 0.9, 1.8, and 3.6 ßg/ml), con-
canavalin A (СопА; 4.5, 9, 18, and 36 дд/ші), pokeweed mitogen
(PWM; 0.6, 1.1, 2.3, and 4.6 ßg/ml), anti-T-cell globulin (ATG;
45, 90, 180, and 360 ßq/ml) , and staphylococcus enterotoxin (STE;
0.0225, 0.045, 0.09, and 0.18 ßg/ml). Cells were cultured for 4
days at 37 0C at a density of 2 χ IO5 cells/well in microtiter
plates in 220 μΐ RPMI 1640 with 0.29 mg/ml L-glutamine, 0.16
mg/ml gentamicin-sulfate, 10% heat-inactivated pooled human
serum, and the above indicated mitogens. For the last 16 h the
cultures were labeled with 2 дСі [3H]thymidine/well. The cells
were harvested with a multiple-sample precipitator (Skatron,
Lier, Norway) and incorporated radioactivity was measured in a
scintillation counter (LKB 1212, Freiburg, FRG).
Drugs and monoclonal antibodies. (-) - [ 125I]ICYP and
(~)-[125I]IPIN (specific activity 2,200 Ci/mmol) , and a radio
immunoassay for cAMP were purchased from Du Pont-New England
Nuclear, and [3H]thymidine (5 Ci/mmol) was from Amersham.
Isoproterenol (Aludrin) was obtained from Boehringer (Ingelheim,
FRG) , and epinephrine (Suprarenin) and norepinephrine (Arterenol)
were from Hoechst (Frankfurt, FRG). Tablets of ICI 118,551 and
bisoprolol were kindly provided by ICI Pharma (Plankstadt, FRG)
and Merck (Darmstadt, FRG), respectively, and (±)-CGP 12177
hydrochloride (4-(3-tertbutylamino-2-hydroxy-
propoxy)benzimidazole-2-one) were provided by Ciba-Geigy (Basel,
Switzerland).
RPMI 1640, FCS, L-glutamine, and gentamicin-sulfate were obtained
from Nunc (Wiesbaden, FRG), and Xylocaine was from Astra Chemicals
(Wedel/Holstein, FRG). PHA (T-cell specific) was from Welcome
(Beckenham, UK), ConA (T-cell specific) was from Sigma Chemical
(Deisenhofen, FRG), PWM (stimulates T- and B-cells) was from
Boehringer, ATG (T-cell specific) was from Fresenius (Oberursel,
FRG), and STE (T-cell dependent) was from Serva (Heidelberg,
FRG). All other chemicals were from Sigma Chemical and of the
highest purity grade available.
42
FITC coupled goat anti-mouse IgG + IgM was purchased from Medac
(Hamburg, FRG). Monoclonal mouse anti-human antibodies were
obtained from the following sources (specificities in
parentheses): 0KB2 (CD24) , 0KT3 (CD3), and 0KT16 (CD7) f rom Ortho
Diagnostic Systems (Neckargemünd, FRG); BI (CD20) from Coulter
Immunology (Krefeld, FRG); Jot3 (CD3), Jot4 (CD4), Jot8 (CD8),
and JotlO (NK-cell marker) from Immunotech Dianova (Hamburg,
FRG); LeuMl (CD15), Leu2 (CD8), Leu3 (CD4), Leu5b (CD2), Leu7
(NK-cell marker), and aDR (class II) from Becton Dickinson
(Heidelberg, FRG); BMA040 (CD4) from Behring (Frankfurt/Main,
FRG); and Tük-1 (CDllb) , TÜ39 (class II), and TÜ102 (CDS) were
kindly provided by Dr. Ziegler (Tübingen, FRG).
Data evaluation. The maximal number of binding sites (Bmax) and
the equilibrium dissociation constant (Kd) for ICYP and IPIN
binding were calculated from plots according to Scatchard (46).
The experimental data given in text, figures, and tables are
means ± SE of л experiments. The significance of differences was
estimated by two-tailed Student's t test; a Ρ value < 0.05 was
considered to be significant.
RESULTS
Isoproterenol infusion in healthy volunteers increased the
ß2-AR density of unfractionated MNL (as determined by binding to
intact cells) dose-dependently with a maximal increase at the
highest dose of - 125-150% (Fig. 1A). Similarly, 10 μΜ
isoproterenol-induced in vitro increases in intracellular cAMP
content were enhanced after isoproterenol infusion (Fig. IB) .
Pretreatment of the volunteers with the ß2-selective AR antagonist
ICI 118,551 markedly shifted the dose response-curve for the
isoproterenol-induced increase in ß2-AR density and cAMP accu
mulation to the right to higher doses, whereas pretreatment with
the ß1-selective AR antagonist bisoprolol did not attenuate the
isoproterenol-induced increase in either parameter, suggesting
a ß2-AR mediated effect (Fig. 1). Infusion of the nonselective
endogenous catecholamine epinephrine also markedly increased the
43
cu
ΙΛ Ol
i/i СП с
•то с
со CL > -
^ОООп
3000·
2000-
1000
Р7
MNL B-Cells (п=Ю) (п=5)
сгэ before ISO
¿д T-Cells Th-CeUs t ^ C e l l s (n=5) (n=5) v c ( n = 5 )
ezaafter ISO
FIG. 6. Ef fects of i s o p r o t e r e n o l (ISO)-infusion on ß2-adrenergic receptor (AR) dens i ty in lymphocyte subse ts in 10 male heal thy vo lun t ee r s . I sopro terenol was infused in doses of 3 .5 , 7, 17.5, 35 and 70 ng k g - 1 min - 1 for 5 mm each; immediately before infusion and a f t e r 25 mm of infusion hepann ized blood was withdrawn for lymphocyte ß2-AR determinat ion in the lymphocyte subse t s . Ordinate : ß2-AR dens i ty in lymphocyte subse t s , determined by Scatchard ana lys i s (46) of I - [125i ) - iodocyanopindolol (ICYP) binding in i n t a c t c e l l s , in ICÏP binding s i t e s / c e l l . Values are means ± SE; η = no. of experiments. x x Ρ < 0.01 vs . T - c e l l s ; * Ρ < 0.05 vs. T h - c e l l s . For a l l lymphocyte subsets ß2-AR dens i ty a f t e r i sopro te reno l infusion was s i g n i f i c a n t l y higher than before i sop ro te reno l infusion (P < 0 .01) . Percent increases in ß2-AR dens i ty amounted to 106.2 ± 10.1 in mononuclear leukocytes (MNL), Θ9.7 ± 9.2 in B-cel l s , 162.4 ± 17.9 in T - c e l l s , 96.5 ± 13.4 in T h - c e l l s , and 77.2 ± 12.5 in T s / c - c e l l s .
ß2-AR dens i ty of c i r c u l a t i n g MNL (as measured on i n t a c t c e l l s , Fig. 2A), whereas the same dose of norepinephrine (38) increased MNL ß2-AR densi ty only marginally (Fig. ЗА).
As ß-AR can e x i s t on the c e l l surface as well as in a sequestered pool, and can migrate from one compartment to another (21, 44), we repeated our experiments with binding to MNL membranes instead of i n t a c t c e l l s . Under these condi t ions i soproterenol infusion a l so e levated the MNL ß2-AR densi ty (Fig. 4A) ; concomitantly b a s a l - as well as i soproterenol -s t imula ted adenylate cyclase a c t i v i t y increased independently of whether expressed in absolute values ( i . e . , pmol cAMP formed -mg prote in - 1 -min - 1 ) or as percent increase above the corresponding basal a c t i v i t y (Fig. 4B) . The
44
EC5o (- 1 ДМ) for the concentration-response curve was not changed
(cf. Fig. 4B) . A 90-inin infusion of epinephrine (Fig. 2B) but
not an equal dose of norepinephrine (Fig. 3B) also increased the
MNL ß2-AR density as determined in membranes.
In contrast to the in vivo effects in vitro incubation of intact
MNL for 60 min at 37 0C with different concentrations isopro
terenol, epinephrine, and norepinephrine resulted in a concen
tration-dependent decrease in MNL ß2-AR density; the rank order
of potency of the catecholamines was isoproterenol > epinephrine
>> norepinephrine (23). In addition, incubation of lymphocytes
obtained before the infusion in autologous plasma obtained
immediately after the isoproterenol infusion for 60 min at 37 0C
did not influence ß2-AR density at all, independently whether
ßj-AR density was determined in intact cells or in membranes
(Fig. 5).
Because it has been demonstrated that MNL subsets have different
numbers of ß-AR (34, 3 6), we determined whether an isoproterenol
infusion might change the subset composition of circulating MNL.
Infusion of isoproterenol did not significantly alter the total
leukocyte counts or the circulating numbers of monocytes and
B-cells (Table 1) but increased the number of peripheral blood
lymphocytes by 26 ± 7% (P < 0.05; Table 1) , resulting in a relative
lymphocytosis. The increased lymphocyte counts resulted from a
slight decrease of Th-cell number (41 ± 3%) and a large increase
of both Tg/C- and NK-cell number (107 ± 18% and 346 + 74%,
respectively; Table 1). Thus, isoproterenol decreased the
Th-/Ts/c-cell ratio from 1.7 to 0.5 (Table 1). Infusion of the
rather ß1-AR selective agonist norepinephrine, however, did not
change the number of any type of circulating leukocytes (Table
2).
We then asked whether this change in the subset composition of
circulating lymphocytes might explain the increase in MNL ß2-AR
density after isoproterenol infusion. Therefore, we measured ß-AR
density in each MNL subtype separately (Fig. 6) . B-cells had
significantly more ß-AR than T-cells (1,779 ±121 vs. 1,047 ± 54
45
ICYP binding sites/cell respectively; Ρ < 0.01), which is in
reasonable agreement with the average number of ß-AR on
unfractionated MNL (1,146 ± 59 ICYP binding sites/cell) and the
proportion of T- and B-cells. Among the T-cells, T3,c-cells had
more ß-AR than Th-cells (1,511 ± 134 vs. 1,086 + 120 ICYP binding
sites/cell, respectively; Ρ < 0.05). In addition, in two
experiments we could determine ß-AR density in NK-cells: it
amounted to - 2,000 ICYP binding sites/cell. The affinity of the
radioligand ICYP for the ß-AR (Kd-value: 16.9 ± 1.6 pM, η = 30),
however did not significantly differ between lymphocyte sub-
populations. Thus an increased relative number of Tg/
c-cells
(which have more ß-AR than Th-cells) could be one reason for the
increase in ß-AR of unfractionated MNL after isoproterenol
infusion.
3000η
¡л гоиН
1000-
>-
Monocytes
(η=5) г 15-| Ш
CL
f io-TD С
è i5' "о 6
ч -nJ
Pio fel ets
In=5)
. . ι
I ι before ISO eaafter ISO
FIG. 7. Effects of isoproterenol (ISO)-infusion on monocyte and platelet
ß2-adrenergic receptor (AR) density in 5 male healthy volunteers. Isoproterenol was infused in doses of 3.5, 7, 17.5, 35 and 70 ng-kg-1-min-1 for 5 min each; immediately before infusion and after 25 min of infusion heparinized blood was withdrawn for determination of monocyte and platelet ß2-AR density. Ordinates: left, monocyte ß2-AR density, determined by Scatchard analysis (46) of i-(125I]-iodocyanopindolol (ICYP) binding in intact cells, in ICYP binding sites/cell; right, platelet ß2-AR density, determined by Scatchard analysis (46) of iodopindolol (IPIN) binding in membranes, in fmol IPIN specifically bound/mg protein. Values are means ± SE; η = no. of experiments.
To determine whether other factors might also participate in
the isoproterenol-induced elevation of MNL ß-AR, we measured
ß2-AR density in each subset of the fractionated MNL after
isoproterenol infusion (Fig. 6). We found that infusion of the
46
ß-AR agonist increased the ß-AR density in B-, pan-T-, Th-, and
TB/c-cells by - 100%, suggesting that the change in MNL subset
distribution does not explain the increase in ß-AR of unfrac-
tionated MNL after isoproterenol infusion. Monocyte and platelet
ß2-AR, however, were not altered by isoproterenol infusion (Fig.
7), suggesting a lymphocyte-specific phenomenon. The affinities
(Kd values) of ICYP (lymphocytes and monocytes) and IPIN (pla
telets) for the ß2-AR were not affected by the isoproterenol
infusion.
2000-1
1500-
^ 100CH
a
? I 500 CD
Q .
ι 1 1 1 1 1
0 15 7 T75 35 70 Isoproterenol (ng/kg/minfor5min each)
FIG. 8. Effects of isoproterenol
infusion on lymphocyte ß2-adrenergic receptor (AR) density in 5 splenec-toroized patients (for details see METHODS). Isoproterenol was infused in doses of 3.5, 7, 17.5, 35 and 70 ng-kg-l min-* for 5 min each; immediately before infusion and after 25 min of infusion hepannized blood was withdrawn for determination of lymphocyte ß2-*R density. Ordinate: lymphocyte ß2-AR density, determined by Scatchard analysis (46) of i-I^SlJ-iodocyanopindolol (ICYP) binding in intact cells, in ICYP binding sites/cell. Given are individual data of 5 patients who had been splenectomized because of Hodgkin's disease (·, 4 patients) or trauma (o, 1 patient). * Means ± SE of ß2-AR density of the 5 patients was significantly (P < 0.05) higher after isoproterenol infusion vs. preinfusion level.
Because the spleen is the largest source of lymphocytes in the
body, we tried to assess the role of this organ in the isoproterenol
infusion-induced changes in subset distribution and ß-AR density
of circulating lymphocytes. For this purpose two patients who
suffered from Hodgkin's disease were infused with isoproterenol
before as well as after splenectomy and two Hodgkin's and one
non-Hodgkin's patient, who was splenectomized because of trauma,
were infused with isoproterenol after splenectomy only. Infusion
of isoproterenol in the two Hodgkin's patients before splenectomy
induced similar changes as in healthy subjects (cf. Fig. 1) :
47
ß2-AR densi ty on unfract ionated MNL increased by - 130% (from 676 t o 1,552 ICYP binding s i t e s / c e l l ) and was accompanied by a r e d i s t r i b u t i o n of lymphocyte subse t s : T s /C- and NK-cell counts increased, whereas T h -ce l l counts decreased (data not shown). Thus the T h - /T s / c - ce l l r a t i o was decreased from 1.5 to 0 .7. After splenectomy, however, i soproterenol infusion increased ß2-AR densi ty on unfract ionated MNL only by - 40% (Fig. 8) and, apart from an increase in NK-cell number, lymphocyte subset composition was not affected (Table 3 ) .
TABLE 3 . Relative distribution of monocytes and lymphocyte subsets in 5 splenectomized patients before and after isoproterenol infusion
Monocytes
B-cells
T-cells
Th-cella
Ts/c-cells
NK-cells
Th" to Ts/C -cell ratio
Isoproterenol Infusion
Before
rel %
11.2±1.9
17.8±3.2
55.8±2.7
34.2±3.1
19.0±3.0
3.6±0.8
1.7±0.3
After
rel %
13.6±3.1
15.6±2.7
52.0±6.0
26.4±3.0
19.6±3.4
8.4±2.4*
1.410.2
Values are means ± SE of 5 experiments. See Table 1 for abbrev ia t ions . I soproterenol was infused in doses of 3 .5 , 7, 17 .5 , 35 and 70 ng kg-^· min -^ for 5 mm each; immediately before infusion and a f t e r 25 min of infusion heparinized blood was withdrawn for determination of d i s t r i b u t i o n of monocytes and lymphocyte subse t s . * Ρ < 0.05 vs. corresponding preinfus ion l e v e l .
As a further measure of functional changes in lymphocyte
responsiveness after infusion of isoproterenol, we determined
the in vitro proliferation of unfractionated MNL (obtained before
and after isoproterenol infusion) in response to various con
centrations of ConA, PHA, ATG, STE, and PWM. After isoproterenol
infusion, the maximal increment in [3H]thymidine uptake was
depressed by - 40-50% on PHA-, ConA-, and ATG-stimulation, 60%
on STE-stimulation, and 70% on PWM-stimulation (Fig. 9) . However,
a slight shift in the optimal stimulatory concentration toward
48
lower concentrations was observed for PHA (Fig. 9). We did not
observe any alteration of the in vitro proliferation after infusion
of norepinephrine (Fig. 10). In splenectomized patients, apart
from a slight depression at 0.6 дд PWM/ml, the in vitro lymphocyte
proliferative response on mitogenic stimulation was not influenced
by isoproterenol infusion (Fig. 11).
120Ί
Οί.5 0.9 1.8 3.6
STE (n=3)
0.6 1.1 2.Э 4.6 Mitogen Concentrahon (μς/ιτί)
ι—I before NE
Е Я after 30 nun NE
ШШ after 60 mm NE
E S after 90 mm NE
00225 QJX5 0u09 (UB
M t o g e n Concentration (pg/ml)
FIG. 10. Effects of norepinephrine (NE) infusion (50 ng kg- 1-min
- 1 for 90
min) on in vitro proliferative response of lymphocytes on mitogen stimulation
in 3 healthy volunteers. See FIG. 9 for abbreviations. Ordinatesi [3H)thymidine
incorporation into lymphocytes in counts/min (cpm) χ 10-3. Abscissas: mitogen
concentrations in μg/ml. Values are means ± SE; л = no. of experiments.
49
izo-
80-
¿
ATG (n^9)
• τ — Ζ
240
160
о
E 80-
О
PHA[n=9)
I **
^
60-
to
zo-
90 ISO
PWM (n=9l
360
Ж ж Ά % QA5 05
60η STE|n=91
UB 3.6
Í.0
ь к
E 20-
а 0.0225
ч
U6 1.1 23 W
Mitogen Concentrahon (μς/ιη()
CD before ISO
E 3 otter ISO
*#
S ¿2 aots 0.09 0.18
Mitogen Concentration (pg/nil)
FIG. 9. Effects of i sopro te reno l (ISO) infusion on in v i t r o p r o l i f e r a t i v e response of lymphocytes on mitogen s t imula t ion in 9 heal thy vo lun tee r s . I sopro te renol was infused in doses of 3 .5 , 7, 17 .5 , 35 and 70 ng k g - 1 min - 1
for 5 min each; immediately before infusion and a f t e r 25 min of infusion hepar inized blood was withdrawn for determination of lymphocyte response t o mitogen s t imu la t i on . ConA, concanavalin A; PHA, phytohemagglutinin; STE, staphylococcus Enterotoxin; ATG, a n t i - T - c e l l g lobu l in ; PWM, pokeweed mitogen. Ord ina tes : [-Ή]thymidine incorporat ion i n t o lymphocytes in counts/min (cpm) x 10 _ 3. Abscissas: mitogen concentrat ions in pg/ml. Values are means ± SE; η = no. of experiments. ** Ρ < 0 .01 , * Ρ < 0.05 vs. t h e corresponding pre infus ion l e v e l s .
DISCUSSION
I t h a s r e p e a t e d l y been r e p o r t e d by s e v e r a l g roups of i n v e s t i g a t o r s
t h a t dynamic e x e r c i s e , menta l s t r e s s , o r exogenous c a t e c h o l a m i n e s
can a c u t e l y i n c r e a s e t h e d e n s i t y of MNL ß-AR (12, 14-17 , 22, 29,
50
0Λ5 0.9 l i 3J6
60-
к Σ. a.
" 20-
0 -
É STE (n=5 )
1
1 1 É
0.6 11 2.3 4.6 Mitogen Concenfrahon [pg/ml)
CD before ISO BZa after ISO
00225 0.045 0.09 0.18 Mitogen Concentration (рд/гЫ)
FIG. 11. Effects of isoproterenol (ISO) infusion on in vitro proliferative
response of lymphocytes on mitogen stimulation in 5 splenectomized patients
(for details see METHODS). Isoproterenol was infused in doses of 3.5, 7, 17.5,
35 and 70 ng kg- 1 m m
- 1 for 5 min each; immediately before infusion and after
25 m m of infusion hepannized blood was withdrawn for determination of
lymphocyte response to mitogen stimulation. See FIG. 9 for abbreviations.
Ordinates: [^HJthymidme incorporation into lymphocytes in counts/min (cpm)
x 10_3. Abscissas: mitogen concentrations in μg/ml. Values are means ± SE; η
= no. of experiments. * Ρ < 0.05 vs. corresponding preinfusion level.
48) . Enhanced cAMP generation in response to isoproterenol
stimulation in vitro after dynamic exercise suggests that the
additional ß-AR are functional(12) . In the present investigation,
we used infusions of adrenergic agonists for a more detailed
analysis of this phenomenon, because this allows a better control
51
of the experimental conditions. With this approach we extend the
earlier observations by showing that the heightened MNL ß-AR
density is found not only when assessed by radioligand binding
to intact cells (Figs. 1 and 2) but also by binding to a plasma
membrane preparation (Figs. 2 and 4) . These data suggest that
the ß-AR increase is not likely to be caused by redistribution
of receptors between cellular compartments. Measurements of
adenylate cyclase activity in a broken cell preparation show an
increased isoproterenol-induced cAMP generation after isopro
terenol infusion. Thus acute catecholamine exposure elevates the
number and responsiveness of MNL ß-AR apparently independent of
the intracellular compartmentation of the receptors. No such
alteration was observed with platelet and isolated monocyte ßj-AR
in vivo, and in vitro exposure of MNL to catecholamines or
autologous plasma obtained after isoproterenol infusion did not
increase ß-AR density. Thus the catecholamine infusion-induced
increase in ß-AR appears to be a lymphocyte-specific effect that
can only be demonstrated in vivo.
Further experiments were designed to characterize pharmaco
logically the receptor mediating the increase in ß-AR number and
responsiveness after acute catecholamine exposure. We have
previously shown that acute pretreatment with propranolol can
block this increase (14) . This and the fact that it can be elicited
by the ß-AR agonist isoproterenol suggest mediation via a ß-AR.
By acutely pretreating our volunteers with either the ß2-selective
antagonist ICI 118,551 or the ß1-selective antagonist bisoprolol
(Fig. 1), and by infusing the nonselective catecholamine epi
nephrine and the rather ßj-selective (38) norepinephrine (Figs.
2 and 3) , we demonstrated that the ß-AR involved is of the
ß2-subtype. This finding is in good agreement with our previous
observation that chronic pretreatment with ICI 118,551, but not
bisoprolol, can also block the isoproterenol-induced increase in
lymphocyte ß2-AR (15).
52
Isoproterenol infusion not only elevated the MNL ß-AR density
but also caused a relative lymphocytosis (Table 1) . A similar
lymphocytosis has been reported after injections of epinephrine
(19) or after dynamic exercise (2, 28). The exercise-induced
lymphocytosis (like the exercise-induced increases in MNL ß-AR)
can be blocked by propranolol (2, 28). However, according to our
data, the number of circulating cells is not equally increased
in all MNL subsets. Whereas the number of total leukocytes,
monocytes, and B-cells remains fairly constant, only the numbers
of circulating T-cells and NK-cells increase. Among the T-cells
Th-cells decrease, but this is more than compensated by an increase
in the number of circulating Ts/c-cells (Table 1) . Thus the
Th-/TB/c-cell ratio decreased. A decreased Th-/Ts/c-cell ratio
after mental stress, dynamic exercise, or injections of
isoproterenol or epinephrine has also been demonstrated by other
investigators (19, 37).
This alteration of the subset composition of circulating
lymphocytes might contribute to the increase in MNL ß-AR, as Ts/C-
and NK-cells have significantly more ß-AR than Th-cells (see Fig.
6 and Ref 34) . However, we demonstrated that isoproterenol infusion
increased the ß-AR density in each subset of circulating lym
phocytes (Fig. 6). Furthermore, a markedly attenuated increase
in the MNL ß-AR density was observed in splenectomized patients
(Fig. 8). We therefore conclude that the increase in MNL ß-AR
after acute isoproterenol infusion is caused by a subset-specific
release of lymphocytes from the spleen into the circulation and/or
by an exchange of lymphocyte subsets between the spleen and the
circulation, whereby freshly released splenic lymphocytes appear
to have more ß-AR than those found in the circulation. The subset
selectivity of this cell-mobilization process can likely be
explained by the fact that different lymphocyte subsets are found
in different regions of the spleen, which differ in their sym
pathetic innervation (for a review see Ref. 27): catecholamines
might influence the migration in (distinct regions of) the spleen
or may act directly at the ß2-AR of specific lymphocyte subsets.
53
The still remaining slight increase in ß-AR density in the
splenectomized patients might be explained by the fact, that
after isoproterenol infusion NK-cells (containing a rather high
number of ß-AR, see above) had significantly increased. Another
possible explanation might be that lymphocytes are mobilized from
tissues other than the spleen (thoracic duct?) but to a lesser
extent.
Two possibilities might account for the higher ß-AR densities
of lymphocytes freshly released from the spleen (or other tissues)
compared with those in the circulation. Circulating (i.e.,
extrasplenic) lymphocytes might be chronically exposed to higher
concentrations of epinephrine, and thus have tonically downre-
gulated ß-AR. This assumption is supported by the evidence that
treatment of healthy subjects and patients with ß-AR antagonists
will upregulate the number of MNL ß-AR (1, 13, 15, 41). A second
possibility arises from the functional differences between splenic
and circulating lymphocytes. Although most circulating lympho
cytes are in a resting state, it is possible that lymphocytes
freshly released from the spleen might have undergone some
preactivation. This idea is supported by the finding that activated
lymphocytes express an enhanced responsiveness of ß-AR compared
with resting lymphocytes (20). This elevated ß-AR responsiveness
should render the circulating lymphocytes more susceptible to
the antiproliferative effects of ß-AR agonists like epinephrine,
and thus would further enhance the impairment of immune function
by stress.
Our investigations of the dose-response relationships between
infused catecholamine and the increase in MNL ß-AR (Fig. 1) and
the previous studies with dynamic exercise or mental arithmetic
stress suggest that the above-described phenomena are not
restricted to our experimental setting but might very well occur
as part of the physiological stress reaction in daily life. To
determine the relevance of such alteration for immune function,
we assessed the in vitro proliferation of peripheral blood
lymphocytes obtained before and after isoproterenol infusion in
54
response to various mitogens (Fig. 9). After infusion of
isoproterenol but not after norepinephrine (Fig. 10) lymphocyte
proliferation in response to each mitogen was reduced by at least
40% (Fig. 9). A reduced mitogenic response to PHA, ConA and PWM
has also been reported after epinephrine injections (18). Thus,
after acute increases in plasma catecholamines (endogenously or
exogenously), immune responsiveness seems to be impaired. This
appears to be a transient effect as lymphocyte mitogen respon
siveness had returned to predrug values 2 h after the injection
of epinephrine (18). Whether the reduced mitogenic response of
the lymphocytes after isoproterenol infusion is because of the
increased ß-AR number or reflects an intrinsic reduction in the
mitogenic responsiveness of the freshly released lymphocytes
remains to be elucidated in further studies.
We conclude that the previously observed increase in MNL ß2-AR
number and responsiveness does not represent receptor regulation
but instead is a lymphocyte specific phenomenon; it is obviously
caused by a release of lymphocyte subsets from secondary lymphoid
organs into the circulation and/or by an exchange of lymphocyte
subsets between secondary lymphoid organs and the circulation,
whereby freshly released lymphocytes appear to carry more ß2-AR
than those found in the circulation. Isoproterenol infusion (and
probably also endogenous acute sympathetic stimulation) appears
to impair immune responsiveness in a dual manner: by increasing
the relative and absolute number of Ts/c-cells, and by making the
circulating T-cells more responsive to inhibitory effects of
ß-adrenergic stimulation in the face of elevated plasma
catecholamines. The effect of chronically elevated sympathetic
activity on immune function remains to be determined.
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60
subtype binding activity in plasma and beta blockade by propranolol
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61
CHAPTER 3. Effects of insulin-induced hypoglycemia on ß2-adre-
noceptor density and proliferative responses of
human lymphocytes
L.J.H. van Tits, A. Daul, H.J. Bauch, H. Grosse-Wilde, M. Happel,
M.C. Michel and O.-E. Brodde
Zentrum für Innere Medizin, Medizinische Klinik und Poliklinik,
Abteilung für Nieren- und Hochdruckkrankheiten, and the Institut
für Immungenetik (H.G.-W., M.H.), Universität Essen, D4300 Essen;
and the Institut für Arterioskleroseforschung an der Universität
Münster (H.J.B.), D-4400 Münster, West Germany
Abstract
We studied the effects of insulin (0.1 lU/kg BW, iv)-induced
hypoglycemia on lymphocyte ß2-adrenoceptor function, lymphocyte
subset distribution, and proliferative response to mitogen
stimulation in 10 healthy volunteers. Thirthy minutes after
insulin injection plasma glucose levels were markedly decreased;
concomitantly, plasma epinephrine levels had increased about
10-fold; plasma norepinephrine levels, however, increased only
moderately. Lymphocyte ß2-adrenoceptor density and the cAMP
response to 10 дтоІ/Ь isoproterenol stimulation were elevated;
lymphocyte Ts/
c-cells had increased, whereas T
h-cells had
decreased, resulting in a decrease in the Th-/T
s/
c-cell ratio
from 1.7 to 1.0. These changes were accompanied by a significantly
reduced lymphocyte proliferative response (measured as
[3H]thymidine uptake) to mitogen stimulation. Two hours after
insulin injection plasma catecholamines, lymphocyte ß2-adreno-
ceptor function, lymphocyte subset distribution, and prolife
rative responses had returned to nearly preinsulin levels. We
conclude that acute vigorous increases in endogenous epinephrine
evoked by insulin-induced hypoglycemia are associated with
increases in lymphocyte ß2-adrenoceptor function, redistribution
of lymphocyte subsets, and an (at least transiently) attenuated
in vitro immune responsiveness.
62
Introduction
Determination of ß-adrenoceptors on circulating mononuclear
leukocytes (MNL) is a frequently used approach to assess
ß-adrenoceptor function in man (1-3). However, this model is of
limited value because of two major reasons. 1) Circulating MNL
contain a homogeneous population of ß2-adrenoceptors; thus,
changes in these receptors might not adequately reflect changes
in ßj-adrenoceptors (4-6). 2) Acute exposure of MNL to cate-
cholaminergic agonists іл vivo causes, in contrast to what has
been observed in all other model systems, not a decrease, but a
rapid increase in ß2-adrenoceptor number (7-11). This is a
ß2-adrenoceptor-mediated effect, as it can be inhibited by the
ß2-adrenoceptor antagonist ICI 118,551, but not by the ß1-adre-
noceptor antagonist bisoprolol (9,10).
On the other hand, evidence has accumulated in man that the
function of the immune system might be influenced by the sym-
patho-adrenal system, possibly via interaction with MNL
ßj-adrenoceptors (12). Thus, in vitro stimulation of MNL
ß2-adrenergic (by isoproterenol) and other adenylate cyclase-
linked receptors can inhibit MNL proliferation, mitogen-induced
secretion of interleukin-2, and expression of interleukin-2
receptors (13-18). ß-Adrenoceptor agonists might also have an
antiproliferative effect in vivo, since we have recently shown
that in healthy volunteers after isoproterenol, but not nore
pinephrine, infusion the proliferative response of circulating
MNL to various mitogens was markedly attenuated (10).
Taken together, these findings suggest effects of
catecholamines on MNL function; however, these effects have
generally been investigated at rather high (nonphysiological)
concentrations of the catecholamines. Whether (patho)physio
logical elevations in plasma catecholamines may have similar
effects on MNL function is still an open question. To answer this
question we have determined in healthy volunteers the effects of
insulin-induced hypoglycemia, known to vigorously elevate plasma
epinephrine levels, on MNL ß2-adrenoceptor number and on the MNL
63
in vitro proliferative response to stimulation with the mitogens
phytohemagglutinin (PHA), Concanavalin-A (Con-A), pokeweed
mitogen (PWM), anti-T-cell globulin (ATG), and Staphylococcus
enterotoxin (STE).
Materials and Methods
•Experimental protocol
Ten healthy volunteers (eight males and two females, aged
25-38 yr; mean age, 30.6 ± 3.1 yr) participated in the study
after having given informed written consent. All subjects were
physicians at the Department of Internal Medicine of the University
of Essen Medical School. They were drug free and had undergone
physical examination to exclude asthma, chronic pulmonary disease,
diabetes mellitus, hypertension, cardiac disease, and other
symptoms referable to the cardiovascular system. At 0800 hour an
indwelling cannula was inserted into a forearm vein. In the
opposite forearm a needle was placed into a large vein and kept
patent with infusion of 0.9% saline. After an initial rest period
of 30 min in the supine position insulin (0.1 lU/kg BW) or 1 mL
0.9% saline was injected iv via the cannula. Immediately before
injection (0 min) and 30, 60, 90, and 120 min after injection
blood samples were withdrawn after first flushing saline from
the infusion needle with about 3 mL blood. For determination of
plasma catecholamines and MNL ß2-adrenoceptors blood was anti-
coagulated with EDTA, for determination of blood glucose levels
with sodium citrate (1 mL 3.2% sodium citrate/10 mL blood), and
for assessment of mononuclear cell function and analysis of MNL
subsets with heparin (500 IU heparin/10 mL blood). Blood pressure
and heart rate were recorded automatically by a Tonomed (Speidel
and Keller, Jungingen, West Germany) and an electrocardiogram.
Cell isolation MNL were prepared from whole blood by Ficoll gradient
centrifugation according to the method of Böyum (19) with minor
modifications (10).
64
Determination of 32-adrenoceptors MNL ß2-adrenoceptors were identified in intact cells by
radioligand binding with (-) -[125I]iodocyanopindolol (ICYP) at
six concentrations of ICYP ranging from 10-150 pmol/L in the
presence and absence of 1 /imol/L (±)CGP 12177 (20). The accu
mulation of cAMP in intact cells during a 15-min incubation in
the absence and presence of 10 μιηοΐ/ΐι isoproterenol was measured
according to previously described techniques (20).
Functional MNL tests
Mononuclear cell function was monitored by the ability of
cells to proliferate upon mitogenic stimulation with PHA (3.6,
1.8, 0.9 and 0.4 5 pg/mL), Con-A (36, 18, 9 and 4.5 ßg/mh), PWM
(4.6, 2.3, 1.14 and 0.6 Mg/mL), ATG (360, 180, 90 and 45 Mg/mL)
and STE (0.18, 0.09, 0.045 and 0.0225 μg/mL). Cells were cultured
for 4 days at 37 "С in microtiter plates containing the mitogens
in 220 μ!. RPMI-1640 with 0.29 mg/mL L-glutamine, 0.16 mg/mL
gentamycin-sulfate, and 10% heat-inactivated pooled human serum
at 2 χ 105 cells/well. For the last 16 h the cultures were labeled
with 2 μ€ί [3H]thymidine/well. The cells were harvested with a
multiple sample precipitator (Skatron, Lier, Norway), and
incorporated radioactivity was assessed in a liquid scintillation
counter (LKB 1212, Freiburg, West Germany).
Analysis of MNL subsets
MNL subsets were analyzed by indirect immunofluorescence
with monoclonal antibodies (OKT3, Jot3 and Leu5b for pan-T cells;
Jot4a, Leu3 and BMA040 for Th-cells; Jot8a, Leu2 and TÜ102 for
Ts/c-cells; TÜ39 and aDR for B-cells; JotlO and Leu7 for NK-cells;
Tük-1 for monocytes; fluorescein isothiocyanate (FITC)-labeled
goat antimouse immunoglobulin G (IgG) plus IgM was used as the
second antibody). In short, MNL were incubated with appropriate
dilutions of monoclonal antibody for 40 min, washed, labeled with
FITC-conjugated goat antimouse antibody for 40 min and washed;
all procedures were performed at room temperature. Cells were
scanned within 2 h under a Zeiss fluorescence microscope (Zeiss,
Oberkochen, West Germany).
65
Plasma catecholamines were assessed by a high performance
liquid chromatographic method with electrochemical detection.
Data evaluation
The maximal number of binding sites for ICYP binding was
calculated from plots according to the method of Scatchard (21) .
The experimental data given in text, figures and the table are
the mean ± SEM of η experiments. Statistical significance of
differences was estimated by two-tailed Student's t-test for
paired data; Ρ < 0.05 was considered significant.
Materials
ICYP (SA, 2200 Ci/mmol) and a RIA for cAMP were purchased
f rom New England Nuclear (Dreieich, West germany) and [3H] thymidine
(5 Ci/mmol) from Amersham (Braunschweig, West Germany). Insulin
(H-Insulin-Hoechst) was obtained from Hoechst (Frankfurt/Main,
West Germany).
(±)CGP 12177 hydrochloride [4-(3-tertiarybutylamino-2-hyd
roxy-propoxy)benzimidazole-2-one] was provided by Ciba-Geigy
(Basel, Switzerland). RPMI-1640, L-glutamine and gentamy-
cin-sulfate were obtained from Nunc (Wiesbaden, West Germany).
PHA was from Welcome (Beckenham, United Kingdom), Con-Α from
Sigma (Deisenhofen, West Germany), PWM from Boehringer (Mannheim,
West Germany), ATG from Fresenius (Oberursel, West Germany), and
STE from Serva (Heidelberg, West Germany). All other chemicals
were from Sigma and of the highest purity grade available.
FITC-coupled goat antimouse IgG plus IgM was purchased from
Medac (Hamburg, West Germany) . Monoclonal antibodies were obtained
from the following sources (specifications in parentheses): 0KT3
(CD3) from Ortho Diagnostic Systems (Neckargemiind, West Germany) ;
Jot3 (CD3), Jot4 (CD4), Jot8 (CD8) and JotlO (NK-cell marker)
from Immunotech Dianova (Hamburg, West Germany); Leu2 (CD8), Leu3
(CD4), Leu5b (CD2), Leu7 (NK-cell marker) and aDR (class II) from
Becton Dickinson (Heidelberg, West Germany), BMA040 (CD4) from
66
Behring (Frankfurt/Main, West Germany); Tük-1 (CDllb), TÜ39 (class
II) and TÜ102 (CD8) were kindly provided by Dr. Ziegler (Tübingen,
West Germany).
Results
Thirty minutes after iv injection of insulin, blood glucose
levels had decreased from 4.27 ± 0.17 to 1.44 + 0.33 mmol/L (Fig.
1). Concomitantly, systolic blood pressure and heart rate
increased significantly, whereas diastolic blood pressure fell
by about 20 mm Hg (Fig. 2) . Plasma epinephrine had increased
almost 10-fold (from 309 ± 82 to 3400 ± 1365 pmol/L); plasma
norepinephrine, however, only moderately increased (from 1.91 ±
0.26 to 2.77 ± 0.45 nmol/L; Fig. 1). During the following 90 min,
blood glucose, plasma catecholamines, and heart rate returned to
predrug levels. Physiological saline solution, on the contrary,
had no significant effect on any of these parameters.
67
о E
50-
4.0-
3.0-
2Û-1.0-
o-l
3.0
ì 20-E с
1.0J
5000 4000-1
^3000
12000-^ Ю О О -
0
Blood Glucose
· — · Insulin
ш-- > NoCl
Plasma Norepinephrine
— -• + -Plasma Epinephrine
Ю
Time (mm)
60 90 720
FIG. 1. Effects of insulin (0.1 lU/kg BW, iv) or 0.9% NaCl on blood glucose,
plasma norepinephrine, and plasma epinephrine levels in 10 healthy volunteers.
Ordinates from top to bottom, Blood glucose in millimoles per L, plasma
norepinephrine levels in nanomoles per L, and plasma epinephrine levels in
picomoles per L. Abscissae, Time after insulin or NaCl injection in minutes.
Given are the mean ± SEM of 10 experiments. **, Ρ < 0.01; *, Ρ < 0.05 (vs.
the corresponding predrug levels).
The effects of insulin-induced hypoglycemia on ß2-adrenoceptors
on MNL are shown in Fig. 3; 30 min after insulin injection (i.e.
at a time when plasma epinephrine levels had markedly increased)
ß2-adrenoceptor density was increased by about 150%. This increase
in receptor density was associated with a similar increase in
the 10 μιηοΙ/L isoproterenol-induced in vitro increase in
intracellular cAMP, indicating that the increase in ß2-adreno-
ceptor number was an increase in real functional receptors. During
the following 90 min, both MNL ß2-adrenoceptor density and cAMP
68
130
^ΙΣΟ
Ι 110-
100 85ι
75· ff ε 65 ε
55
US-90·
80-
ί 70
1 боі
A. Systolic Blood Pressure
50J
«--« NoCl · — · Insulin
f <-
В DJostolic Blood Pressure
С Heart Rate
30 Time (mm)
TO" 90 Тго
FIG. 2. Effects of i n s u l i n (O.l lU/kg BW, iv) or 0.9% NaCl on blood pressure and hear t r a t e in 10 heal thy v o l u n t e e r s . Ordmates from top to bottom, S y s t o l i c and d i a s t o l i c blood pressure in mm Hg, hear t r a t e in beats per min. Abscissae, Time a f t e r i n s u l i n or NaCl i n j e c t i o n in minutes . Given are t h e mean ± SEM of 10 experiments. ** , Ρ < 0.01 vs. the corresponding predrug l e v e l s .
r e s p o n s e t o 10 μιηοΐ/ΐ, i s o p r o t e r e n o l r e t u r n e d t o p r e d r u g l e v e l s
( F i g . 3) , a s d i d plasma c a t e c h o l a m i n e s (cf . F i g . 1) . P h y s i o l o g i c a l
s a l i n e s o l u t i o n , on t h e o t h e r hand, had no s i g n i f i c a n t e f f e c t on
MNL ß2 - ad renocep to r d e n s i t y or t h e 10 μιηοΙ/L i s o p r o t e r e n o l - i n d u c e d
i n v i t r o i n c r e a s e i n CAMP ( F i g . 3 ) .
69
Anj-Adrcnoceptor Density
BWumol/l ISO-evoked cyclic AMP Increase
Time (mm)
FIG. 3. Effects of insulin (0.1 lU/kg BW, iv) or 0.9% NaCl on MNL ß2_adrenc>cePtoi:' density and 10 |jmol/L isoproterenol (ISO)-evoked in vitro cAMP increase in five healthy volunteers. Ordinates, upper panel, MNL ß2-adrenoceptor density, determined by Scatchard analysis (21) of ІСУР binding in intact cells, in
ICYP-binding sites per cell; lower panel, 10 μπιοΐ/ί isoproterenol-evoked m vitro increase m MNL cAMP content in picomoles cAMP per 10
6 cells. Abscissae,
Time after insulin or NaCl injection in minutes. Given are the mean ± SEM of
five experiments. **, Ρ < 0.01; *, Ρ < 0.05 (vs. the corresponding predrug
levels).
Analysis of MNL subsets revealed that, apart from a slight
increase in B-cells 30 min postinjection, there were no changes
in the relative percentages of total T-lymphocytes, B-lymphocytes,
or monocytes after insulin administration (Table 1). The per
centage of Τ,,-cells, however, declined from 35.0 before insulin
injection to 25.6 and 28.8 30 and 60 min postinjection,
respectively, and returned to baseline by 120 min. On the contrary,
the percentage of Ts/
c-cells increased from 21.8 before insulin
injection to 27.4 and 28.0 30 and 60 min postinjection,
respectively, and returned to nearly baseline by 120 min (Table
70
1) . Accordingly, the T h -/T B / c -cel l r a t i o decreased from 1.7 before
i n j e c t i o n t o 1.0 and 1.1 30 and 60 min post inj e e t ion, r e s p e c t i v e l y ,
and then increased to 1.5 120 min pos t in jec t ion (Table 1) . The
percentage of NK-cells increased from a basel ine of 4.0 t o 6.6
and 6.0 30 and 60 min p o s t i n j e c t i o n , re spect ive ly , and decl ined
t o 4.8 120 min p o s t i n j e c t i o n (Table 1) .
TABLE 1. Re la t ive d i s t r i b u t i o n of monocytes and lymphocyte subsets in f ive
healthy sub ject s before and a f t e r i n s u l i n i n j e c t i o n (0.1 lU/kg BW)
Monocytes
B-cells
T-cells
Th-cells
Ts/
c-cells
NK-cells
Th-to-TB/
c cell ratio
Before
relative %
5.211.3
14.6±2.0
64.2+2.8
35.0±2.7
21.8±2.6
4.0±1.0
1.7+0.3
After insulin
30 min
relative %
5.2+1.5
16.8±2.1a
62.212.9
25.6+2.9b
27.4+3.5
6.6il.3a
i.oio.Ia
60 min
relative %
6.012.0
13.611.9
65.013.5
28.8i2.3b
28.0i2.2b
6.0+1.0b
1.1+0.Ia
120 min
relative %
4.010.9
17.013.1
64.512.9
34.3+1.5
24.312.7
4.811.1
1.5+0.2
Given are t h e mean 1 SEM of five experiments. a Ρ < 0.05 vs. t h e corresponding p r e i n s u l i n l e v e l s . k Ρ < 0.01 vs. t h e corresponding p r e i n s u l i n l e v e l s .
Finally we assessed the effects of insulin-induced hypoglycemia
on mononuclear cell function. For this purpose we measured the
proliferative in vitro responses of unfractionated MNL to various
concentrations of the mitogens PHA, Con-Α, PWM, ATG, and STE. As
shown in Fig. 4, 30 and 60 min after insulin injection the maximal
[3H]thymidine incorporation into MNL was significantly reduced
upon stimulation with all mitogens investigated, whereas 120 min
after insulin injection the responses had returned to nearly
predrug levels.
Discussion
Tohmeh and Cryer (7) were the first to report that short
term infusion of isoproterenol or epinephrine into healthy
71
CON A |n .51 АТС 1пз5І
06 1.1 2.3 44 Mitogen Concentration (до'тО
'—' before Inwlin s o otter 30mm Insulin mm atitrtCmm Insulin ^ s at ter 120 m η Insulin
aaZ75 0045 009 (UB
Mitogen Concentration (uj/ml)
FIG. 4. Effects of insulxn (0.1 lU/kg BW, iv) or 0.9% NaCl on the m vitro proliferative response of MNL to mitogen stimulation in five healthy volunteers.
Ordinates, (ЗщтЬуткЗте incorporation into MNL in counts per min κ ΙΟ-·';
abscissae, mitogen concentrations in micrograms per mL. Given are the mean ±
SEM of five experiments. **, Ρ < 0.01; *, Ρ < 0.05 (vs. the corresponding
predrug levels).
volunteers causes an increase in the density of MNL ß2-adreno-
ceptors. We recently showed that this isoproterenol-induced
increase in MNL ß2-adrenoceptor density can be antagonized by
the selective ß2-adrenoceptor antagonist ICI 118,551, but not by
the selective ß-^adrenoeeptor antagonist bisoprolol, demon
strating that the ß-adrenoeeptor involved in this process is of
the ß2-subtype (9,10). This conclusion is further supported by
the fact that infusion of epinephrine, but not of an equal dose
of (the rather ß^^-selective) norepinephrine caused similar
increases in MNL ß2-adrenoceptors (10).
72
In the present study we wanted to determine whether such
changes in MNL ß2-adrenoceptor function can be achieved not only
by exogenously applied ß-adrenoceptor agonists used in rather
high (nonphysiological) doses but also by (patho)physiological
elevations of endogenous catecholamines. For this purpose we have
used insulin-induced hypoglycemia, since this is known to be the
most powerful stimulus to rapidly release epinephrine from the
adrenal medulla. In fact, the marked decrease in plasma glucose
levels 30 min after iv injection of insulin was associated with
an about 10-fold increase in plasma epinephrine, but an only
modest increase in plasma norepinephrine. The vigorous increase
in plasma epinephrine was accompanied by about a 150% increase
in MNL ß2-adrenoceptor number and MNL in vitro cAMP response to
10 ßmol/h isoproterenol stimulation, increases comparable with
(or higher than) those evoked by infusion of exogenous epinephrine
or isoproterenol (10). A similar rapid acute increase in MNL
ß2-adrenoceptors associated with rapid elevation of plasma
epinephrine levels has recently been also observed in a patient
with pheochromocytoma during a hypertensive crisis (22) . Taken
together, these data strongly suggest that not only exogenously
applied catecholamines, but also (patho)physiological increases
in endogenous epinephrine can evoke marked increases in MNL
ß2-adrenoceptor function.
The rapid increase in MNL ß2-adrenoceptor density after
acute ß-adrenergic stimulation with isoproterenol or epinephrine
appears to be a lymphocyte-specific effect, since 1) it is not
seen for ß2-adrenoceptors on monocytes or platelets (10), and 2)
it has been demonstrated in rats that 15 min after isoproterenol
injection pulmonary ß-adrenoceptor density is decreased (23) . We
have recently shown that infusion of isoproterenol not only
acutely increases MNL ß2-adrenoceptor density, but also causes
a redistribution in MNL subsets (10). Thus, after isoproterenol
infusion the percentages of Te/C- and NK-cells increased, whereas
that of Th-cells decreased, resulting in a reduced Th-/Ts/c-cell
ratio. As shown in Table 1, very similar changes in lymphocyte
subsets occurred during insulin-induced hypoglycemia; 30 and 60
73
min after insulin application (i.e. at a time when plasma epi
nephrine levels were elevated), the relative amount of Th-cells
was decreased, and that of Tg/C- and NK-cells was increased,
resulting in a diminished Th-/Ts /c-cell ratio. A decreased
Th-/Ts/c-cell ratio after insulin-induced hypoglycemia as well
as after epinephrine injection has also been demonstrated by
other investigators (24-26) .
As Tg/C- and NK-cells have nearly twice as many ß-adrenoceptors
as Th-cells (10,27,28), such a redistribution may well contribute
to the rapid increase in ß2-adrenoceptor density in unfractionated
MNL during insulin-induced hypoglycemia. However, redistribution
of lymphocyte subsets cannot completely explain the marked
increase in MNL ß2-adrenoceptor density observed in the present
study because 1) the increase is much higher than could be achieved
assuming that it is only caused by redistribution, and 2) we
recently demonstrated that after isoproterenol infusion (which
increased MNL ß2-adrenoceptors to a similar extent) ß-adrenoceptor
density in each subset had increased as well. On the other hand,
in splenectomized patients the increase in MNL ß2-adrenoceptors
after isoproterenol infusion was markedly blunted (10). Taken
together, these results indicate that the marked increase in MNL
ß2-adrenoceptor density after infusion of high (nonphysiological)
doses of epinephrine or isoproterenol or accompanying the vigorous
(patho)physiological increases in plasma epinephrine evoked by
insulin-induced hypoglycemia is due to subset-specific release
of lymphocytes from the spleen into the circulation; the lym
phocytes obtained from peripheral blood after this vigorous acute
catecholamine exposure obviously contain more ß-adrenoceptors
than those present in the circulation before the catecholamine
exposure (10).
In the present study the insulin-induced acute increase in
plasma epinephrine was not only accompanied by heightened MNL
ß2-adrenoceptor density and redistribution of MNL subsets, but
also by an attenuated proliferative response of MNL to various
mitogens, as could be expected from the decrease in the
74
Th-/T
8/
c-cell ratio. Again, this effect is in very good agreement
with our recent observation that after isoproterenol infusion
into healthy volunteers the Th-/T
g/rc-cell ratio is decreased, and
the mitogenic response of MNL to PHA, Con-Α, ATG, STE, and PWM
is reduced (10). As the present results show, this appears to
be, however, a transient effect; 30 and 60 min after insulin
injection, when plasma epinephrine levels are markedly elevated,
the mitogenic response was reduced, while after 120 min, when
epinephrine levels had decreased to predrug values, the mitogenic
response had nearly returned to predrug response.
In conclusion, the present results demonstrate that acute
increases in catecholamines (regardless of whether exogenously
applied or endogenously elevated) generally lead to an increase
in MNL ß2-adrenoceptor function. These changes are accompanied
by a (spleen-dependent) redistribution of lymphocyte subsets and
an (at least transiently) attenuated іл vitro immune respon
siveness. Whether such transient changes in circulating lymphocyte
subsets have any effect on immune responsiveness in vivo, however,
remains to be elucidated in future experiments. Hypoglycemia is
a commonly occurring phenomenon in insulin-dependent diabetic
patients. Although the immunological parameter studied is only
a crude assessment of immunocompetence (lymphocyte enumeration)
and is of uncertain significance in regard to disease sus
ceptibility, it might contribute to the frequent infections in
insulin-dependent diabetic patients or other patients with acutely
or chronically heightened sympathetic activity. Further under
standing of the interaction between the sympatho-adrenal and the
immune system may enable the development of new therapeutic
approaches for immunological and inflammatory disorders in man.
References
1. Motulsky HJ and Insel PA. Adrenergic receptors in man. Direct
identification, physiologic regulation, and clinical alterations.
N Engl J Med. 1982;307:18-29.
2. Brodde O-Ε, Beckeringh JJ, Michel MC. Human heart ß-adre-
noceptors: a fair comparison with lymphocyte ß-adrenoceptors?
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Trends Pharmacol Sci. 1987;8:403-7.
3. Brodde O-Ε, Wang XL. Beta-adrenoceptor changes in blood
lymphocytes and altered drug responsiveness. Ann Clin Res.
1988;20:311-23.
4. Michel MC, Pingsmann A, Beckeringh JJ, Zerkowski Η-R, Doetsch
N, Brodde O-Ε. Selective regulation of ϋγ- and ß2-adrenoceptors
in the human heart by chronic ß-adrenoceptor antagonist treatment.
Br J Pharmacol. 1988;94:685-692.
5. Liggett SB, Marker JC, Shah SD, Roper CL, Cryer PE. Direct
relationship between mononuclear leukocyte and lung ß-adrenergic
receptors and apparent reciprocal regulation of extravascular,
but not intravascular, a- and ß-adrenergic receptors by the
sympathochromaf f in system in humans. J Clin Invest. 1988;82:48-56.
6. Michel MC, Pingsmann A, Nohlen M, Siekmann U, Brodde O-E.
Decreased myometrial ß-adrenoceptors in women receiving
ß2-adrenergic tocolytic therapy: correlation with lymphocyte
ß-adrenoceptors. Clin Pharmacol Ther. 1989;45:1-8.
7. Tohmeh JF, Cryer PE. Biphasic adrenergic modulation of
ß-adrenergic receptors in man. Agonist-induced early increment
and late decrement in ß-adrenergic receptor number. J Clin Invest.
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8. DeBlasi A, Maisel AS, Feldman RD, Ziegler MG, Fratelli M,
DiLallo M, Smith DA, Lai C-YC, Motulsky HJ. In vivo regulation
of ß-adrenergic receptors on human mononuclear leucocytes:
assessment of receptor number, location, and function after
posture change, exercise, and isoproterenol infusion. J Clin
Endocrinol Metab. 1986;63:847-53.
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JJ. Differentiation of ß 1- and ß2-adrenoceptor-mediated effects
in humans. Am J Physiol. 1988:254:H199-206.
10. Van Tits LJH, Michel MC, Grosse-Wilde H, Happel M, Eigler
F-W, Soliman A, Brodde O-Ε. Catecholamines increase lymphocyte
ß2-adrenergic receptors via a ß2-adrenergic, spleen-dependent
process. Am J Physiol. 1990;258:E191-202.
11. Brodde O-Ε, Daul A, Michel-Reher M, Boomsma F, Man in't Veld
AJ, Schlieper P, Michel MC. Agonist-induced desensitization of
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ß-adrenoceptor function in man. Subtype-selective reduction in
ßj- or ß2-adrenoceptor-mediated physiological effects by xamoterol
or procaterol. Circulation. 1990;81:914-21.
12. Feiten DL, Feiten SY, Bellinger DL, Carlson SL, Ackerman KD,
Madden KS, Olschowki JA, Livnat S. Noradrenergic sympathetic
neural interactions with the immune system: structure and
function. Immunol Rev. 1987;100:225-60.
13. Sanders VM, Munson AE. Beta-adrenoceptor mediation of the
enhancing effect of norepinephrine on the murine primary antibody
response in vitro. J Pharmacol Exp Ther. 1984;230:183-92.
14. Hellstrand К, Hermodsson S, Strannegard Ö. Evidence for a
ß-adrenoceptor-mediated regulation of human natural killer cells.
J Immunol. 1985;134:4095-9.
15. Bourne HR, Lichtenstein LM, Melmon KL, Henney CS, Weinstein
Y, Shearer GM. Modulation of inflammation and immunity by cAMP:
receptors for vasoactive hormones and mediators of inflammation
regulate many leucocyte functions. Science. 1974;184:19-29.
16. Feldman RD, Hunninghake GW, McArdle WL. ß-Adrenergic
receptor-mediated suppression of interleukin 2 receptors in human
lymphocytes. J Immunol. 1987;139:3355-9.
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The effects of autacoids on cloned murine lymphoid cells:
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18. Kammer GM. The adenylate cyclase cAMP-protein kinase A pathway
and regulation of immune response. Immunol Today. 1988;9:222-8.
19. Böyum A. Isolation of mononuclear cells and granulocytes from
human blood. Scand J Clin Lab Invest. 1968;21 (Suppl 97):77-89.
20. Brodde 0-E, Brinkmann M, Schemuth R, O'Hara N, Daul A.
Terbutaline-induced desensitization of human lymphocyte
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21. Scatchard G. The attraction of proteins for small molecules
and ions. Ann NY Acad Sci. 1949;51:660-672.
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Acute regulation of lymphocyte ß-adrenoceptor activity in
pheochromocytoma. Klin Wochenschr. 1988;66:187-9.
23. Strasser RH, Stiles GL, Lefkowitz RJ. Translocation and
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catecholamine promoted desensitization in vivo. Endocrinol.
1984;1392-400.
24. Frier BM, Corral RJM, Davidson NM, Webber RG, Dewar A, French
EB. Peripheral blood cell changes in response to acute hypoglycemia
in man. Eur J Clin Invest. 1983;13:33-9.
25. Fisher BM, Smith JG, McCruden DC, Frier BM. Responses of
peripheral blood cells and lymphocyte subpopulations to insu
lin-induced hypoglycemia in human insulin-dependent (type 1)
diabetes. J Immunol. 1983;130:694-7.
26. Crary B, Borysenko M, Sutherland DC, Kutz I, Borysenko JZ,
Benson H. Decrease in mitogen responsiveness of mononuclear cells
from peripheral blood after epinephrine administration in humans.
J Immunol. 1983;130:694-697.
27. Khan MM, Sansoni Ρ, Silverman ED, Engleman EG, Melmon KL.
ß-Adrenergic receptors on human suppressor, helper, and cytolytic
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78
CHAPTER 4. Cyclic ΑΗΡ counteracts mitogen-induced inositol
phosphate generation and increases in intracellular
Ca2+-concentrations in human lymphocytes
"Lambertus J. H. van Tits, "'Martin С. Michel, 'Harvey J. Motulsky,
**Alan S. Maisel & *Otto-Erich Brodde
*Biochem. Research Labs, Dept. Medicine, Div. Renal & Hypertensive
Diseases, University of Essen, D-4300 Essen, F.R.G. *Dept. of
Pharmacology, M-036, University of California San Diego, La Jolla,
CA 92093 and "Cardiology Section 111-A, Veterans Administration
Medical Center, San Diego, CA 92161, U.S.A.
Keywords: mononuclear leukocytes, cyclic AMP, inositol phos
phates, calcium, human lymphocytes, immunoregulation
1) The effects of increases in intracellular adenosine
3',5'-cyclic monophosphate (cyclic AMP) on mitogen-induced
generation of inositol phosphates and increases in intracellular
Ca2 + concentration were investigated in human peripheral blood
mononuclear leukocytes (MNL).
2) The mitogens concanavalin A (Con A ) , pokeweed mitogen (PWM)
and phytohemagglutinin (PHA) concentration-dependently stimu
lated generation of inositol phosphates. Catecholamines inhibited
this process with an order of potency: isoprenaline > adrenaline
> noradrenaline indicating involvement of ß2-adrenoceptors. This
order of potency was also consistent with the catecholamine
potencies for stimulating the generation of cyclic AMP.
3) In addition to catecholamines the cyclic AMP formation-sti
mulating agents prostaglandin Ει (PGE^ and forskolin concen
tration-dependently inhibited mitogen-induced inositol phosphate
generation, too. Moreover, the inhibitory effect of isoprenaline
was potentiated by co-incubation with the phosphodiesterase
inhibitor isobutylmethylxanthine demonstrating that these
inhibitory effects were mediated by cyclic AMP.
79
4) Con A and PHA concentration-dependently increased the
intracellular Ca2 + concentration in human MNL (assessed by the
fluorescent indicator dye Fura-2). This increase was almost
completely blocked by chelation of extracellular Ca++, demon
strating influx rather than mobilization from intracellular
stores.
5) The elevation of intracellular Ca2+ was not blocked by
pre-treatment with pertussis toxin, 100 ng ml- 1 for 16 h.
6) Isoprenaline, PGE^ and forskolin, however, inhibited the
mitogen-stimulated elevation of intracellular Ca2+. This inhi
bition was enhanced by the phosphodiesterase inhibitors isobu-
tylmethylxanthine and Ro 20-1724, demonstrating mediation by
cyclic AMP.
7) We conclude that catecholamines and other cyclic AMP increasing
agents can inhibit mitogen-stimulated generation of inositol
phosphates and elevation of intracellular Ca2 + in resting human
MNL. Since generation of inositol phosphates and increases in
intracellular Ca2+ are very early events in activation of MNL,
attenuation of these events might well contribute to the inhibitory
effect of cyclic AMP on MNL function.
Introduction
The function of the immune system is regulated by various
neurotransmitters and hormones. Of special interest are those
agents which elevate lymphocyte adenosine 3',5'-cyclic mono
phosphate (cyclic AMP) concentrations because they can inhibit
lymphocyte function in multiple ways іл vitro (for reviews see
Bourne et al., 1974; Feiten et al., 1987; Kammer, 1988). For
example, it has been demonstrated that cyclic AMP increasing
hormones can inhibit mitogen-stimulated lymphocyte proliferation,
antibody formation by B-cells, and cytolytic activity of T-cells.
These inhibitory effects of cyclic AMP formation stimulating
hormones are mediated by cyclic AMP because they can also be
elicited by receptor-independent stimulation of cyclic AMP
formation (e.g. by the diterpene forskolin) and by compounds
80
mimicking cyclic AMP (e.g. 8-Br-cyclic AMP). Moreover, some data
suggest that cyclic AMP increasing agents can also impair immune
function in vivo. For example it has been shown that prolonged
exposure to ß-adrenoceptor agonists can decrease the number of
thymocytes in mice (Durant, 1986) and of circulating lymphocytes
in man (Maisel et al., 1990).
The activation of resting lymphocytes is a multistep process
involving early biochemical alterations such as formation of
inositol phosphates and increases in intracellular Ca2+ leading
to activation of a protein kinase C, which occur during the first
seconds and minutes of lymphocyte activation (Tsien et al., 1982;
Wolff & Akerman, 1982; Taylor et al., 1984, Sugiura Ь Waku, 1984;
Chen et al., 1986; Cambier et al., 1987; Hadden, 1988). These
early alterations are followed within a couple of hours by later
events such as secretion of interleukin-2 (11-2), and expression
of 11-2 receptors and certain oncogenes. This second wave of
biochemical events can also be inhibited by elevations of
intracellular cyclic AMP (Feldman et al., 1987; Mary et al.,
1987; Johnson et al., 1988; Kammer, 1988). As secretion of 11-2
and expression of 11-2 receptors depend on an elevation of
intracellular Ca2+ and an activation of a protein kinase С (Cambier
et al., 1987; Hadden, 1988), it is possible that cyclic AMP
formation stimulating agents inhibit lymphocyte activation by
attenuating the generation of these early biochemical signals.
Therefore, we have investigated the effects of various cyclic
AMP formation stimulating agents on the mitogen-stimulated
generation of inositol phosphates and increases of intracellular
Ca2* in resting human mononuclear leukocytes (MNL, mostly lym
phocytes) , with special emphasis on the effects of ß-adrenoceptor
agonists.
Methods
Mononuclear leukocyte preparation
Mononuclear leukocytes (MNL), containing >95% lymphocytes, were
prepared from the venous blood of healthy young volunteers
according to the method of Böyum (1968). In some experiments,
81
MNL were cultured in RPMI medium supplemented with 20% foetal
calf serum, 2 mM glutamine and 25 ßg ml-1 gentamycin in the absence
or presence of 100 ng ml-1 pertussis toxin for 16-20 h prior to
the Ca2+ measurements. These cells were washed twice in buffer 2
h before the Ca2+ measurements in order to remove the pertussis
toxin.
Cyclic AMP measurements
Accumulation of cyclic AMP was measured in intact MNL according
to previously published techniques (Brodde et al. , 1985) . Briefly,
MNL were suspended in phosphate buffered saline containing 0.1
mM theophylline (in order to inhibit cyclic AMP degradation by
phosphodiesterases) and 0.025% bovine serum albumin. Aliquots of
300 μΐ (2xl06 cells) were incubated for 15 min at 37
0C in the
presence or absence of indicated stimuli, in a total volume of
330 μΐ. Incubation was terminated by boiling for 5 min. After
cooling, samples were centrifugea at 12,000 g for 10 min, and
cyclic AMP content was determined in 50 μΐ aliquots of the
supernatant by radioimmunoassay.
In some experiments MNL were preincubated with mitogens for
15 min at 370C before theophylline (0.1 mM final concentration)
and isoprenaline were added. Incubations were continued for
another 15 min, and then stopped and samples processed as described
above.
Inositol phosphate generation
Generation of inositol phosphates was determined according to
published techniques (Minneman & Johnson, 1984) with minor
modifications. MNL were suspended in Krebs-Henseleit solution
containing (mM) : NaCl 118, KCl 4.7, CaClj 1.3, MgS04 1.2, KH
2P0
4
1.2, NaHC03 24.9, glucose·H2O 11, EDTA 1 μΜ and 0.1% bovine serum
albumin, equilibrated with 95% 02/5% COj at 37 0C. Cells were
incubated with [3H]-myo-inositol (10 μΟί ml
- 1 with 3xl0
7 cells
ml-1) for 2 hours at 37
0C. Thereafter, the cells were washed and
resuspended in Krebs-Henseleit solution supplemented with 10 mM
LiCl (to block inositol phosphate breakdown). Aliquots of this
cell suspension (450 μΐ containing 5xl06 cells) were preincubated
82
for 15 min under 95% 02/ЪЧ COj; thereafter 50 μΐ of drug solution
(mitogens in the presence or absence of cyclic AMP formation
stimulating agents) was added. Samples were incubated for another
30 min at 37 0C, and the reaction was stopped by addition of 1.5
ml chloroform-methanol (1:2, v:v). Each sample was mixed tho
roughly 4 times in a period of 1 hour, 0.5 ml of chloroform and
0.5 ml of water added sequentially, mixed again twice and
centrifuged at 3000 g for 12 min to separate the phases. Columns
containing 1 ml of a 1:1 slurry of Dowex AG 1-X8 anion exchange
resin (200-400 mesh) in the formate form, previously stored in
0.1 M formic acid, were washed twice with 10 ml of 10 mM Tris-formate
(pH 7.4) and once with 7 ml water. Aliquots of 1.5 ml of the
upper phase of each sample were filled up to 5 ml with water and
poured on the columns. After washing the columns with 5 ml of
water and twice with 5 ml of 25 mM ammonium formate successively
(to elute remaining [3H]-inositol), the [
3H]-inositol phosphates
retained on the columns were eluted with 1 ml of 2 M ammonium
formate dissolved in 0.1 M formic acid; thus the eluate contained
a mixture of inositolmonophosphate, -bisphosphate and -tris-
phosphate in which inositolmonophosphate is the major constituent
(accumulating from breakdown of the other phosphates in the
presence of LiCl). Scintillation fluid (9 ml) was added to the
eluent and the samples were counted in a Beekman LS 9000 liquid
scintillation counter (Beekman Inst., Fullerton, CA) at an
efficiency of about 42%. Following each experiment, the columns
were regenerated with 2 ml of 2 M ammonium formate dissolved in
0.1 M formic acid and 2 ml of 1 M formic acid, washed with 10 ml
of 10 mM Tris-formate (pH 7.4) and stored in 0.1 M formic acid.
With this regeneration procedure, the columns could be used for
up to 2 months without any loss of capacity.
Ca2 + measurements
Intracellular Ca2 + concentrations were measured with the fluor
escent dye Fura-2 according to standard procedures previously
described in detail (Motulsky ft Michel, 1988) . Briefly, loading
of the cells with Fura-2 and the subsequent fluorescence
measurements were performed at room temperature in buffer con-
83
taining (mM) : NaCl 120, KH2P04 5, Мд(СНзСОО)2 l, СаС1
2 1, Hepes
20 and glucose 1 mg ml- 1 at pH 7.4. Our calculations to convert
raw fluorescence data into intracellular Ca2 + concentrations
included correction factors for dilution of cells by addition of
drugs (1-2%), and for the presence of extracellular Fura-2
(accounting for 10-25% of the total fluorescence), and (in some
experiments) the presence of EGTA. Experiments at 37eC yielded
qualitatively similar but more variable results as more dye leaked
out of the cells; this dye leakage was attenuated by addition of
2.5 mM probenecid but even under those conditions it accounted
for at least 50% of total fluorescence after an incubation period
of more than 10 min (data not shown).
Chemicals
Myo-[2-3H(N)]-inositol (specific activity 20 Ci mmol
-1) and a
radioimmunoassay for cAMP were purchased from New England Nuclear
(Dreiech, FRG) and Amersham (Braunschweich, FRG), respectively.
ICI 118,551 (erythro-(±)-1-(7-methylindan-4-yloxy)-3-isopropyl
aminobutan-2-ol) was a gift from ICI-Pharma (Plankstadt, FRG),
CGP 20712A (l-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3
-[4-(l-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propan
ol methanesulphonate) from Ciba Geigy (Basel, Switzerland), Ro
20-1724 (4-(3,4-dibutoxybenzyl)-2-imidazolidinone) from
Hoffman-La Roche (Basel, Switzerland), and nifedipine from Pfizer
(New York, N.Y., U.S.A.). Dowex AG 1-X8 anion exchange resin
(200-400 mesh, formate form) was obtained from Bio-Rad Labora
tories (Richmond, VA, U.S.A.), Fura-2 acetoxymethoxyester was
obtained from Molecular Probes (Eugene, OR, U.S.A.), forskolin
was from Calbiochem (La Jolla, CA, U.S.A.), and pertussis toxin
from List (Campbell, CA, U.S.A.). Phytohaemagglutinin (PHA) for
the experiments on cyclic AMP and inositol phosphate generation
was from Welcome (Beckenham, U.K.), and the PHA for the Ca2"1-
experiments was from Sigma (St Louis, MO, U.S.A.); these two
preparations differ with respect to their purity grades and,
therefore, their potencies. Pokeweed mitogen (PWM) was from
84
Boehringer (Mannheim, FRG), concanavalin A (Con A) and all other
Chemicals were from Sigma and of the highest commercially available
purity grade.
Data analysis
Maximum and the half-maximal effective concentrations of agonists
were calculated from concentration-response curves by fitting
the experimental data to a sigmoid curve by computer-assisted
non-linear regression analysis. To obtain optimal estimates for
the maximal effect and the concentration of agonist causing 50%
of the maximal effect (EC50) , the Hill-slope and the bottom of
the curve were fixed at 1 and the measured basal value,
respectively, during the curve fitting procedure. Data are shown
as mean ± s.e.mean of η experiments. Differences between groups
were compared with two-tailed t-tests as appropriate with Ρ <
0.05 considered statistically significant.
Results
Cyclic AMP accumulation
Isoprenaline, adrenaline, and noradrenaline concentration-de-
pendently increased the intracellular cyclic AMP content in human
MNL (Table 1) . Although all three catecholamines elicited a
similar maximal cyclic AMP accumulation, their potencies differed
considerably with a rank order of isoprenaline > adrenaline >
noradrenaline. Prostaglandin E^ (PGEj) and forskolin also
stimulated cyclic AMP accumulation in MNL with maximal stimulatory
effects 4-5 and 0.66 times as large as those in response to
isoprenaline, respectively (Table 1) .
As other investigators have reported enhanced hormone-stimulated
cyclic AMP accumulation in the presence of mitogenic lectins
(Fredholm et al., 1987; Dailey et al., 1988), we also determined
isoprenaline-stimulated cyclic AMP accumulation following
exposure to mitogens for 30 min. This protocol was chosen to
yield a mitogen exposure of the MNL comparable to that during
the inositol phosphate accumulation experiments. However, neither
85
Table 1: Concentration-response relationships for Catecholamine-, Prosta
glandin Εχ- and Forskolin- stimulated c y c l i c AMP generation in human lym
phocytes.
Isoprenaline
Adrenaline
Noradrenaline
Forskolin
Prostaglandin Εχ
ECso (nM)
7.8+1.4
48±12
5100±29OO
31OOO±7OO0
1560±800
Maximal cyclic AMP accumu
lation
(pmol/106 cells in 15 min)
47.6±10.6
49.4±8.6
47.5±12.3
31.4±10.5
249±60
Lymphocytes were incubated for 15 min a t 370C with 5-7 c o n c e n t r a t i o n s of t h e i n d i c a t e d a g o n i s t s in t h e presence of 0.1 mM t h e o p h y l l i n e , and t h e c y c l i c AMP content was determined as described in methods. The agonis t concentra t ion causing a half-maximal increase in cyc l ic AMP content , t h e maximal increase and t h e H i l l s lope were c a l c u l a t e d for each experiment by f i t t i n g the experimental data t o a sigmoid curve using computer a s s i s t e d non-l inear regres s ion a n a l y s i s . The H i l l slopes did not d i f f e r s i g n i f i c a n t l y from 1.0. Each value i s t h e mean ± SEM of 4 experiments.
PWM (2 .4 ßg ml - 1 ) nor PHA (1 .8 дд m l - 1 ) s i g n i f i c a n t l y a l t e r e d t h e
p o t e n c y o r maximal e f f e c t s of i s o p r e n a l i n e on MNL c y c l i c AMP
a c c u m u l a t i o n ( F i g u r e 1 ) .
I n o s i t o l phosphate accumulation
I n c u b a t i o n of MNL, p r e l a b e l l e d w i t h [ 3 H ] - m y o - i n o s i t o l , w i t h
v a r i o u s c o n c e n t r a t i o n s of PHA ( 0 . 2 - 1 4 . 4 ßg m l - 1 ) , Con A (0 .14-36
ßg ml - 1 ) and PWM ( 0 . 1 5 - 4 . 8 ßg ml'1) fo r 30 min a t 370C concen-
t r a t i o n - d e p e n d e n t l y i n c r e a s e d t h e fo rmat ion of [ 3 H ] - i n o s i t o l
p h o s p h a t e s ( F i g u r e 2 ) ; t h e maximal e f f e c t was h i g h e s t f o r Con A
and PHA and lowes t f o r PWM. Al l f u r t h e r e x p e r i m e n t s were performed
u s i n g 1.8 ßg ml"1 PHA, 18 ßg m l - 1 Con A, and 2 .4 ßg ml"1 PWM.
86
[Isoprenalme], м
Figure 1 I soprenal ine-induced c y c l i c AMP generat ion in the absence and presence of pokeweed mitogen(PWM) and phytohaemagglutinin (PHA). Lymphocytes were preincubated for 15 min without (contro l ) or with PHA (1.8 jjg m l - l ) or PWM (2.4 μg ml~l) p r i o r t o addi t ion of i s o p r e n a l i n e . Values are mean of 4 experiments for c o n t r o l (open c i r c l e s ; max. c y c l i c AMP generat ion 45.3±7.8 pmol/106 c e l l s in 15 min), PHA ( f i l l e d diamonds; 50.415.9 pmol/106 c e l l s in 15 min) and PWM ( c r o s s e s ; 47.6±4.9 pmol/10^ c e l l s in 15 min); v e r t i c a l bars show s.e.mean. Mean data were f i t t e d t o a sigmoid curve.
ß-Adrenoceptor a g o n i s t s d e c r e a s e d m i t o g e n - s t i m u l a t e d i n o s i t o l
phospha te g e n e r a t i o n by 30-50% depending on t h e mitogen used
(F igu re 3 a - c ) . The maximal i n h i b i t o r y e f f e c t was s i m i l a r f o r a l l
t h r e e c a t e c h o l a m i n e s bu t t h e i r po tency d i f f e r e d , t h e rank o r d e r
b e i n g : i s o p r e n a l i n e > a d r e n a l i n e > n o r a d r e n a l i n e (F igu re 3 ) . The
c a t e c h o l a m i n e - i n d u c e d i n h i b i t i o n of t h e i n o s i t o l phospha t e
g e n e r a t i o n was comple te ly b locked by 100 nM of t h e ß2 -ad renocep to r
s e l e c t i v e a n t a g o n i s t ICI 118,551 ( B i l s k i e t al., 1983) bu t was
no t a f f e c t e d by 500 nM of t h e ßj^-adrenoceptor s e l e c t i v e a n t a g o n i s t
CGP 20712A (Dooley e t a l . , 1986; F i g u r e 4 a - c ) .
87
« M (О
ν к. υ с "5 E 'й α E
100
75
50
25
04l-Œ= m-*
4000
Χ
ζ I
Maximal formation
10" ΙΟ"3 ю-2 Ю - 1
(Mitogen), mg ml"'
Figure 2 Concentration-response curves for mitogen-induced accumulation of
inositol phosphates in human lymphocytes. The EC50 values for pokeweed mitogen
(PWM, crosses), phytohaemagglutinin (PHA, open circles) and concanavalin A
(Con A, filled diamonds) were 0.6 μg ml~l, 1.3 μg ml-!,
a n cj 2.3 μg ml
-^,
respectively. Inset: Maximal Inositol phosphate generation by mitogens
expressed as c.p.m. Data are mean of 3-5 experiments; vertical bars show
s.e.mean.
88
a PHA
100
90
Θ0
70
60
50
к ï— • Vi • \ \
\
•v^T
Nr
vi ÏT X
1 ·
i»
m α V) о .с α
b Con А
100
90
SDI
TO
60 \
50
- с PWM
100
90
Θ0
70
60
50
•
Κ 1
• NN • \
ί.
^
x \
ì\ V ifcs*
,
ν \
—I ^ 2 ,
г S t- - . ? г
IO"9 ю-8 10 7 IO"6 IO"5 10"
[Drug], м
Figure 3 Inhibition of mitogen-induced inositol phosphate generation by
isoprenaline (open circles), adrenaline (filled circles) and noradrenaline
(crosses) in human lymphocytes: (a) phytohaemagglutinin (PHA); (b) concanavalin
A (Con A) and (c) pokeweed mitogen (PWM) . Mean data were fitted to a competition
89
a PHA
с о m Ώ E и υ η
-С
а
α о <л о с
с о
с
Isoprenalme Adrenaline Noradrenaline
40
30
20
10
0
b Con A
.
Λ •
Is op
*
I m е п і Isoprenaline Adrenaline Noradrenaline
50
40
30
20
10
0 10
le PWM
•
•
'
J-
##
i Κ ί Λ Μ
Isoprenaline Adrenaline Noradrenaline
Figure 4 Effects of the ßi-adrenoceptor selective antagonist CGP 20712A (500 nM, filled columns) and the ß2-adrenoceptor selective antagonist ICI 118,551 (100 nM, hatched columns) on catecholamine-evoked inhibition (open columns) of mitogen-induced inositol phosphate generation in human lymphocytes: (a) phytohaemagglutinin (PHA); (b) concanavalin A (Con A) and (c) pokeweed mitogen (PWM). Data are expressed as % inhibition of the formation of [3H)-inositol phosphates caused by the mitogens m the respective experiments. Values are mean of 3-6 experimente; vertical bars show s.e.mean. * P<0.05; ** P<0.01 vs. control.
90
In order to investigate whether the inhibitory effects of the
catecholamines are related to their stimulation of cyclic AMP
generation, we studied the effects of PGE^ and forskolin (which
stimulate cyclic AMP generation independently of ß-adrenoceptor
activation) and of the phosphodiesterase inhibitor isobutylme-
thylxanthine (IBMX). Forskolin, a poor stimulator of cyclic AMP
generation in resting human MNL (see Table 1) , weakly reduced
mitogen-stimulated inositol phosphate generation (20-35% inhi
bition; Figure 5a-c). In contrast, PGE1 (which stimulates cyclic
AMP generation considerably more than the catecholamines, see
Table 1) inhibited mitogen-stimulated inositol phosphate accu
mulation to a much larger extent than the catecholamines (Figure
5a-c). Moreover, IBMX (0.1 mM) considerably enhanced the
inhibition of mitogen-stimulated inositol phosphate generation
by isoprenaline (Figure 6) . Thus, inhibition of inositol phosphate
generation by these various cyclic AMP promoting agents was
strongly related to the increase in the intracellular level of
cyclic AMP.
о о 100
ω J5 О з
.£E оЗ
S 8
•s <»
•§& — о
*
75
50
25
PHA Con A PWM
Figure 6 Effects of the phosphodiesterase inhibitor isobutylmethylxanthine
(0.1 mM, hatched columns) on isoprenaline-evoked inhibition of mitogen-induced
inositol phosphate generation (open columns) in human lymphocytes. Mitogens:
phytohaemagglutinin (PHA); concanavalin A (Con A) and pokeweed mitogen (PWM).
Data are expressed as % inhibition of formation of [3H]-inositol phosphates
caused by the mitogens in the respective experiments. Values are mean of 5
experiments; vertical bars show s.e.mean. *** P<0.001 vs. control in the
absence of isobutylmethylxanthine.
91
• PHA
α ел О CL
к»
75
50
25
0
•
Ν
.
- * — I -
ч о-.
" • • Ν
^
Ч
1 _ . _
Ь Con А
ico
75
50
25
О
0 \
. ч
loo
75
50
25
0
с PWM
i •
о
ю-8 io ' ю-8 i o 5
(PGE, or forskolin], м
10-
Figure 5 Inhibition of mitogen-induced inositol phosphate generation in human
lymphocytes by prostaglandin Ej (open circles) and forskolin (filled circles):
(a) phytohaemagglutinin (PHA); (b) concanavalin A (Con A) and (c) pokeweed
mitogen (PWM). Values are mean of 3-5 experiments: vertical bars show s.e.mean.
92
Increases of intracellular Ca2* concentrations The basal intracellular Ca
2 + concentration was 62 ± 5 nM (n=12
with 8-22 replicates in each experiment) . Con A and PHA con-
centration-dependently elevated intracellular Ca2 + to a peak level
within 1-3 min; as expected, the Ca2 + concentration later declined
from the peak to a plateau above the basal level which was
maintained for more than 30 min (data not shown). From the peak
Ca2 + concentration elicited by 4 mitogen concentrations we con
structed concentration-response curves (Figure 7 ) . PWM elicited
only very small and inconsistent increases of intracellular Ca2 +
(maximally 25 nM at 96 дд ml- 1) regardless of whether experiments
were performed at 250C or ЗУС (data not shown) . Chelation of
extracellular Ca2 + by addition of 5 nM EGTA immediately prior to
the mitogens completely abolished the Ca2 + increases stimulated
by Con A and PHA (n=4; data not shown) . Thus, the mitogen-induced
increases in intracellular Ca2 + were predominantly due to influx
rather than intracellular mobilization in accordance with recently
reported data in human and murine resting lymphocytes (Alcover
et al., 1987; Linch et al., 1987; Gelfand., 1987).
75-
c о
25
PGE, Forskolin Isoprenaline
Figure 8 Inhibition of phytohaemagglutinin (PHA, open columns) and concanavalin
A (Con A, hatched columns) -induced Ca2 + increases in human lymphocytes by
prostaglandin Ej (PGEi, 10 μΜ), forskolin (20 μΜ) or isoprenaline (10 μΜ).
Values are mean of 4-7 experiments; vertical bars show s.e.mean. The mito
gen-induced rise in intracellular Ca2 + in these experiments was 119 ± 17 nM
for PHA and 94 ± В nM for Con A. * P<0.05; *** P<0.001 vs. control in the
absence of cyclic AMP increasing agents.
93
100
0) en «α
« 5 75 с Я)
^ 50 Я)
ε ' χ (О
E о
а?
25-
ISO 2 с « и S 100 υ с
if 60 α E к β
2 0
Τ
< χ α.
Τ
< с ο υ
0 Ч Н -ιο- 10" 10-1
[Mitogen], mg ml- 1
Figure 7 Mitogen-stimulated increases of intracellular Ca2+ in human lym
phocytes. The EC50 values for phytohaemagglutinin (PHA, open circles) and
concanavalin A (Con A, filled circles) were 22.8 μg ml-l and 65.5 μ9 ml~l,
respectively. Inset: Maximal Ca^+ increase expressed in nM. Data are mean of
7 experiments; vertical bars show 9.e.mean.
Overnight pretreatment of MNL cells with pertussis toxin (100
ng ml-1) ADP-ribosylated more than 90% of the pertussis toxin
substrates of these cells, but did not inhibit the mito
gen-stimulated Ca2 + increases (data not shown) . Such a lack of
effect of pertussis toxin to inhibit Ca2+ increases has previously
also been reported in human and murine T-lymphocytes (Macintyre
et al., 1988; Aussei et al., 1988; Gray et al., 1989; Stewart et
al., 1989).
The cyclic AMP generation-stimulating agents PCE1 (10 μΜ) ,
forskolin (20 μΜ) , and isoprenaline (10 μΜ) did not significantly
alter the intracellular Ca2+ concentration of resting MNL (2 ±
2 nM for PGEj, 1 ± 0 nM for forskolin, and 1 ± 1 nM for isoprenaline,
л=4-8 each). However, all three agents inhibited the PHA- and
Con Α-stimulated Ca2+ elevations (Figure 8). Inhibition of the
Ca2 + elevation was associated with a prolonged time-to-peak (data
not shown) which was not quantified in detail. Inhibition of
94
PWM-stimulated Ca2+ increases was not tested because these
increases were too small and inconsistent to begin with (see
above). Blocking the degradation of cyclic AMP with the phos
phodiesterase inhibitors IBMX (0.1 mM) and Ro 20-1724 (0.25 mM)
enhanced the inhibitory effects of PGE^ forskolin, and
isoprenaline (Figure 9) , demonstrating that the inhibition is
mediated by cyclic AMP.
a PHA
PGE, Forskolin Isoprenaline
Figure 9 Effect of phosphodiesterase inhibition by isobutylmethylxanthine
and Ro 20-1724 (100 and 250 μΗ, respectively) on mitogen-stimulated Ca2 +
increases in human lymphocytes. Either the phosphodiesterase inhibitors
(hatched columns) or vehicle (open columns) were added to the cells, followed
20-30 s later by prostaglandin Εχ (1 μΜ), forskolin (2 μΜ), or isoprenaline
(10 μΜ), and 20-30 s later by phytohaemagglutinin (PHA, 100 μς ml- 1, a) or
concanavalin A (Con A, 100 μg ml-l, b) . Data shown are mean of 4-6 experiments;
vertical bars show s.e.mean. * P<0.05 vs. inhibition of Ca2+ increase in the
absence of phosphodiesterase inhibitors.
Discussion
95
The activation of T-lymphocytes is a multistep process. Two of
the first signals which can be observed biochemically are an
elevation of the free intracellular Ca2* concentration and the
hydrolysis of phosphatidyl-inositol-bisphosphate into
inositol-(l,4,5)-trisphosphate (IP3) and diacylglycerol (DAG)
leading to activation of a protein kinase С (Chen et al., 1986;
Cambier et al., 1987; Linch et al., 1987; Gelfand et al., 1987;
Hadden, 1988; Gupta, 1989). Although generation of IP3 is the
major stimulus to elevate intracellular Ca2+ in many model systems
(Berridge & Irvine, 1989) it is still a matter of debate whether
Ca2 + increases in lymphocytes by mitogenic lectins are caused by
IP3 or not (King, 1988) . Our study did not directly address this
question, but the observation that PWM was approximately half as
effective as Con A in generating inositol phosphates but a very
poor stimulus for Ca2 + elevations, support the idea that Ca
2+
influx in lymphocytes may not necessarily be predominantly caused
by inositol phosphate generation.
In our study we have used the T-lymphocyte specific mitogenic
lectins PHA and Con A and the non-specific B- and T-lymphocyte
mitogen PWM to mimic the activation of resting lymphocytes by
antigens. The characteristics of inositol phosphate generation
and elevation of intracellular Ca2+ in human MNL as stimulated
by PHA, Con A and PWM in our and other studies (i.e. concen
tration-dependency; dependency of Ca2+ increase on influx; lack
of pertussis toxin sensitivity) are very similar to those described
previously for other polyclonal T-lymphocyte activators such as
monoclonal anti-CD3 antibodies (for review see Chen et al. , 1986;
Cambier et al., 1987; Hadden, 1988). Therefore, this paradigm is
likely to give reliable estimates of the biochemical alterations
occurring in resting lymphocytes undergoing activation by
antigens. On the other hand, it should be noted that the mitogenic
lectins used in this study may utilize (at least partially)
different pathways for lymphocyte activation.
96
Various authors have shown that cyclic AMP can play an inhibitory
role in the activation of resting T-lymphocytes. Thus, increases
in the intracellular level of cyclic AMP inhibit mitogen- or
antigen-induced T-cell proliferation, cytotoxic T-lymphocyte
function, lymphokine secretion, and production and effects of
11-2 (Bourne et al., 1974; Feiten et al., 1987; Feldman et al.,
1987; Mary et al., 1987; Kammer, 1988; Johnson et al., 1988).
Whether this inhibitory effect of cyclic AMP, however, is due to
interaction with the phospholipase C/IP3/DAG pathway, is still
a matter of debate. Imboden et al., (1986) and Sommermeyer &
Resch (1990) found in the human Τ cell tumour line Jurkat that
cholera toxin (which via ADP-ribosylation activates the adenylate
cyclase coupled Gs-protein thereby stimulating cyclic AMP for
mation) inhibits antigen-induced ІРз-formation and increases in
intracellular Ca2+, but this effect could not be mimicked by other
cyclic AMP increasing agents and/or cyclic AMP analogues. On the
other hand. Lerner et al., (1988) in mouse T-lymphocytes and
Windebank et al., (1988) in cloned human NK cell lines could
demonstrate that increases in the intracellular level of cyclic
AMP, regardless of whether it was receptor-dependent (PGEjJ or
-independent (forskolin), evoked inhibition of IP3 formation and
increases in intracellular Ca2+.
These data prompted us to investigate in mixed human lymphocytes
the effects of various agents known to stimulate the formation
of cyclic AMP on the mitogen-induced generation of inositol
phosphates (as a parameter of phosphatidyl-inositol-bisphosphate
hydrolysis) and increases in intracellular Ca2+. The results
clearly demonstrate that all agents used in this study that
elevate cyclic AMP levels (see Table 1) also inhibited mito
gen-induced generation of inositol phosphates and increases in
the intracellular Ca2 + concentration.
The fact, that (i) inhibition of the break-down of cyclic AMP
by phosphodiesterase inhibitors potentiated the inhibition of
inositol phosphate generation and of intracellular Ca2+ rises and
(ii) the amount of inhibition of inositol phosphate generation
97
was strongly related to the magnitude of cyclic AMP elevations
is in favour of the idea that cyclic AMP is responsible for
inducing these inhibitory effects. Thus, the present data obtained
in human MNL confirm those of Lerner et al., (1988) obtained in
mouse T-lymphocytes and of Windebank et al. (1988) obtained in
cloned human NK cells that cyclic AMP-induced inhibition of cell
proliferation and cytotoxicity, respectively, is accompanied
(and/or induced?) by inhibition of increases in the intracellular
Ca2+ concentrations and generation of inositol phosphates.
In the present study, ß-adrenoceptor agonists increased the
intracellular cyclic AMP content and inhibited the mitogen-induced
generation of inositol phosphates with an order of potency:
isoprenaline > adrenaline > noradrenaline. This order of potency
(Lands et al., 1967) and the fact that catecholamine-induced
inhibition of inositol phosphate generation could be abolished
by the ß2-adrenoceptor selective antagonist ICI 118,551 (Bilski
et al., 1983) but not by the ßj^-adrenoceptor selective antagonist
CGP 20712A (Dooley et al., 1986) strongly support the view that
this effect is mediated via ß2-adrenoceptor stimulation, as is
to be expected since human MNL contain exclusively ß2-adreno-
ceptors (for review see Brodde et al., 1987).
It should be noted that cyclic AMP (a) inhibited inositol
phosphate generation and Ca2+ increases, although both responses
may occur independently; (b) inhibited the second messenger
generation by all three mitogens, although they may stimulate
second messenger generation via at least partially different
pathways (see above); and (c) inhibited second messenger gene
ration independently whether near maximally effective (Con A and
PWM) or low (PHA) mitogen concentrations were used. Thus, our
data suggest a prominent role of cyclic AMP-increasing agents in
the regulation of the generation of second messengers which are
important in the activation of resting lymphocytes.
Taken together our data demonstrate that catecholamines can
attenuate the generation of two second messengers which are
important in the activation of resting human lymphocytes. This
98
appears to be mediated via ßj-adrenoceptors by elevating
intracellular cyclic AMP. Thus, attenuation of early second
messenger responses involved in the activation of resting lym
phocytes may represent (or at least participate in) the molecular
basis for the immunomodulatory effects of the sympathetic nervous
system.
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Macintyre, E.A., Tatham, P.E.R., Abdul-Gaffar, R. and Linch,
D.C. (1988) . The effects of pertussis toxin on human Τ lymphocytes.
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Maisel, A.S., Knowlton, K.U., Fowler, P., Rearden, Α.,
Ziegler, M.G., Motulsky, H.J., Insel, P.A. and Michel, M.C.
(1990). Adrenergic control of circulating lymphocyte subpopu
lations. Effects of congestive heart failure, dynamic exercise,
and terbutaline treatment. J . Clin. Invest. 85: 462-467.
101
Mary, D. , Aussei, С , Ferrua, В. and Fehlmann, M. (1987).
Regulation of interleukin 2 synthesis by cAMP in human Τ cells.
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Minneman, K.P. and Johnson, R.D. (1984). Characterization of
alpha-1 adrenergic receptors linked to [3H]-inositol metabolism
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Motulsky, H.J. and Michel, M.C. (1988). Neuropeptide Y
mobilizes Ca2+ and inhibits adenylate cyclase in human erythro-
leukemia cells. Am. J. Physiol. 255: E880-E885.
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Stewart, S.J., Prpic, V, Johns, J.Α., Powers, F.S., Graber,
S.E., Forbes, J.T. and Exton, J.H. (1989). Bacterial toxins affect
early events of Τ lymphocyte activation. J^ Clin. Invest. 83 :
234-242.
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Moore, J.P. (1984) . Mitogens increase phosphorylation of phos-
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102
CHAPTER 5. Adrenergic involvement in control of lymphocyte
immune function
ABSTRACT
Sympathetic involvement in control of immune function has been
demonstrated by effects of catecholamines (and other compounds
which elevate intracellular cAMP content) on immune parameters
in vitro, ex vivo, and in vivo. In the present study, we examined
the effects of ß-blocker treatments on parameters of cellular
immune function.
9 Day treatments with the ß-blockers bisoprolol, Celiprolol or
propranolol had neither effect on in vitro lymphocyte prolif
eration, nor on circulating lymphocyte numbers, indicating that
the basal sympathetic tone does not play an important role in
control of cellular immune function via ß2-adrenoceptors.
Acute activation of the sympathetic nervous system by infusion
of isoprenaline, was accompanied by a redistribution of circu
lating lymphocyte numbers, causing a decrease in the helper to
suppressor/cytolytic -cell ratio. Neither bisoprolol or
Celiprolol nor propranolol, independently whether given acutely
or chronically, blocked the redistribution of circulating lym
phocytes. Accordingly, this effect is either not ß-adrenoceptor
mediated, or might involve an atypical ß-adrenoceptor. Infusion
of isoprenaline also caused a depression of in vitro lymphocyte
proliferative responses. This depression, however, could be
prevented by treatment with propranolol but not bisoprolol or
Celiprolol, indicating involvement of the ß2-adrenoceptor in the
effect. Accordingly, the phenotypical profile (e.g. the helper
to suppressor/cytolytic -cell ratio) is not responsible for the
depressed lymphocyte in vitro proliferation. More likely, lym
phocytes possessing altered intrinsic properties, and released
into the circulation via stimulation of ß2-adrenoceptors, are
responsible for the reduced proliferative capacity.
103
It is concluded that chronic ß-blocker therapy might be expected
to cause no major changes in cellular immune function. However,
in states of short-term elevated sympathetic activity, ß-blocker
therapy with propranolol prevents a depression of lymphocyte
proliferative capacity.
INTRODUCTION
Evidence is accumulating that the sympathetic nervous system
participates in the control of the immune system. Sympathetic
nerve endings densely innervate lymphoid tissues (for a review
see 1) and lymphoid cells have membrane receptors of the
ß2-adrenergic type for the sympathetic neurotransmitters (2) . In
vitro, stimulation of these ß-adrenoceptors has been shown to
inhibit lymphocyte proliferation and several steps preceding
proliferation (for a review see 3). Moreover, we have recently
shown inhibitory effects of catecholamines in the nanomolar range
on the earliest events following mitogenic activation of lym
phocytes, namely the generation of inositol phosphates and an
elevation of intracellular calcium (4).
Functional evidence for influence on immune function by the
sympathetic nervous system іл vivo concerns changes in the numbers
of circulating lymphocytes. Acute increases in the activity of
the sympathetic nervous system and concomitant increases in
endogenous plasma catecholamines by exercise (5,6,7) or by
insulin-induced hypoglycemia (8), or acute stimulation of
ß-adrenoceptors by infusion of isoprenaline (9) or epinephrine
(10) , cause a relative lymphocytosis with increases in the numbers
of suppressor/cytolytic T-cells and natural killer cells and a
decrease in the number of helper T-cells.
The aim of the present study was to determine influences of the
basal sympathetic tone on cellular immune function. For this
purpose the effects of a 9 day treatment of healthy volunteers
with the ß-blockers bisoprolol (ß1-selective without intrinsic
sympathomimetic activity (ISA)), Celiprolol (ßi-selective with
ß2-ISA) and propranolol (non-selective without ISA) on basal
104
lymphocyte subset composition and in vitro proliferative responses
of lymphocytes to various mitogenic agents were investigated. In
addition the effects of ß-blocker treatment on isoprenaline
infusion-induced changes in lymphocyte subset composition and in
vitro proliferative responses were studied.
METHODS
.Experimental protocol.
28 Healthy male volunteers (age 28.7 ±2.7 (25-34) yr) participated
in the study after having given informed written consent. All
volunteers were drug free for at least 3 wk before the study,
and had undergone physical and electrocardiogram examination to
exclude signs of cardiovascular and pulmonary diseases.
Volunteers were treated with ß-adrenoceptor antagonists for 9
days. 8 Volunteers received propranolol (4x40 mg/day), a
non-selective ß-antagonist without intrinsic sympathomimetic
activity; 8 volunteers received bisoprolol (1x10 mg/day), a
ßj^-selective antagonist without intrinsic sympathomimetic
activity (11); and 7 volunteers received Celiprolol (1x200
mg/day) , a ßj^-selective antagonist with ß2-selective intrinsic
sympathomimetic activity (11). The doses of the ß-blockers were
chosen as such because they have previously been shown to be
effective in modulating lymphocyte ß2-adrenoceptor number (11)
or in antagonizing ßj-adrenoceptor mediated effects (12). The
last intake of bisoprolol and Celiprolol was at 1900 h in the
evening before and for propranolol at 0600 h in the morning of
the isoprenaline infusion.
Additional isoprenaline infusion tests were performed in five
male volunteers (age 26.3 ± 1.8 yr) who received propranolol
intravenously (Dociton, 5 mg) 45 minutes before the test.
After an initial rest period of 1 h in the supine position,
isoprenaline (seguential doses of 3.5, 7, 17.5, 35 and 70
ng.kg~1.min"1 for 5 min each) was infused in a large peripheral
fore-arm vein. Blood pressure and heart rate were recorded
automatically by a Tonomed (Speidel & Keller, Jungingen, FRG)
105
and an electrocardiogram. Immediately before and after infusion,
30 ml peripheral venous blood was withdrawn from the opposite
fore-arm with 500 IU heparin/10 ml blood.
Cell isolation. Peripheral blood lymphocytes were prepared from whole heparinized
blood by Ficoll gradient centrifugation according to the method
of Böyum (13) with minor modifications (9).
Lymphocyte proliferation. Lymphocyte function was monitored by the ability of cells to
proliferate upon mitogenic stimulation with phytohemagglutinin
(PHA; 1.8 ßg/ml) , concanavalin A (ConA; 9 /ig/ml) , pokeweed mitogen
(PWM; 4.6 дд/ті), anti-T-cell globulin (ATG; 360 дд/ті) and
staphylococcus enterotoxin (STE; 0.09 дд/ті). These mitogen
concentrations had been shown to maximally stimulate uptake of
3H-thymidine (9) . Cells were cultured for 4 days at 37
0C in
microtiter plates, containing the mitogens in 220 μΐ RPMI 1640
with 0.29 mg/ml L-glutamine, 0.16 mg/ml gentamycin-sulfate and
10% heat inactivated pooled human serum at 2 χ 105 cells per
well. For the last 16 hours the cultures were labeled with 2 дСі
3H-thymidine per well. The cells were harvested with a multiple
sample precipitator (O. Hiller, Madison USA) and incorporated
radioactivity was assessed in a liquid scintillation counter
(TRI-CARB, Packard).
Analysis of lymphocyte subsets.
Lymphocytes were analyzed by indirect immunofluorescence with
the following monoclonal antibodies: Jot3 for pan-T cells; Jot4a
and BMA040 for helper T-cells (Th); Jot8a and BMA080 for sup-
pressor/cytolytic T-cells (Tg/C) ; aDR for B-cells; JotlO for
natural killer cells (NK). LeuMl was used for quantification of
monocytes and granulocytes present in the cell suspensions.
FITC-labeled goat anti-mouse IgG + IgM was used as the second
antibody. In short, lymphocytes were incubated with appropriate
dilutions of monoclonal antibody for 40 min, washed, labeled with
106
FITC-conjugated goat anti-mouse antibody for 40 min and washed;
all procedures were performed at room temperature. Cells were
analyzed within 2 hours under a Zeiss fluorescence microscope.
Materials.
Isoprenaline (Aludrin) was obtained from Boehringer (Ingelheim,
FRG). Tablets of bisoprolol and Celiprolol were kindly provided
by Merck (Darmstadt, FRG) and Woelm Pharma, respectively. Dociton
and tablets of propranolol were obtained from ICI Pharma
(Plankstadt, FRG).
[3H]-thymidine (5 Ci/mmol) was purchased from Amersham.
RPMI 1640, L-glutamine and gentamycin-sulfate were obtained from
Nunc (Wiesbaden, FRG). PHA was from Welcome (Beckenham, U.K.),
Con A from Sigma (Deisenhofen, FRG) , PWM from Boehringer (Mannheim,
FRG), ATG from Fresenius (Oberursel, FRG), and STE from Serva
(Heidelberg, FRG). All other chemicals were from Sigma and of
the highest purity grade available.
FITC coupled goat anti-mouse IgG + IgM was purchased from Medac
(Hamburg, FRG). Monoclonal antibodies were obtained from the
following sources (specificities in parentheses): Jot3 (CD3),
Jot4a (CD4) , JotSa (CD8) and JotlO (NK-cell marker) from Immunotech
Dianova (Hamburg, FRG), LeuMl (pan-monocyte/granulocyte marker)
and aDR (class II) from Becton Dickinson (Heidelberg, FRG), BMA040
(CD4) and BMA080 (CD8) from Behring (Frankfurt/Main, FRG).
Data evaluation.
All data are shown as means ± SEM of η experiments. Differences
between groups were compared with two-tailed paired t-test.
RESULTS
Effects of ß-blocker treatments on basal lymphocyte subset composition. Pan T-, helper T-, suppressor/cytolytic T-, pan B-, and natural
killer cells were monitored. The number of granulocytes and
monocytes present in the lymphocyte suspensions did not exceed
107
5% and was similar for all preparations. Treatment with a
ß-adrenoceptor antagonist for 9 days did not influence basal
lymphocyte subset composition (Fig 1) .
£ÈÂ
ÂÀ
NK
Figure 1. Isoprenaline infusion-induced changes in circulating lymphocyte subsets of healthy volunteers under control conditions and after treatment with the ß-blockers bisoprolol (n=8), Celiprolol (n=7) and propranolol (n=8). For each subset the first two columns represent pre-treatment values before and after infusion of isoprenaline, respectively, and the thirth and fourth column represent post-treatment values before and after infusion of isoprenaline, respectively. Shown are means ± SEM of η subjects. * P<0.05, P<0.01
vs. the corresponding pre-infusion value.
Effects of ß-blocker treatments on isoprenaline infusion-induced
changes in lymphocyte subset composition.
Infusion of isoprenaline did not change the relative amounts of
pan T-cells and pan B-cells. However, within the population of
T-cells, the percentage of helper cells decreased (39.1 ± 1.0
before vs. 27.6 ± 1.2 after infusion, n=23, p<0.0001) and of
3 о LJ
сл O)
о
(Ό
о
о
bisoprolal
рап-Т т./ pan-β
1 0 8
suppressor/cytolytic cells increased (25.9 ± 1.0 before vs. 31.8
± 0.9 after infusion, n=23 , p=0.0003). Accordingly, Thel_
per/Tsuppressor/cytoiyti.c -cell ratio decreased from 1.57 ± 0.07 to
0.89 ± 0.05 (n=23, p<0.0001) . The percentage of NK cells increased
from 5.1±0. 8 to 11.0 ± 1.4 (n=23, p=0.0007) following isoprenaline
infusion.
A 9 day treatment with bisoprolol or Celiprolol did not influence
the isoprenaline infusion-induced changes in lymphocyte subset
composition (Fig 1,2). Treatment with propranolol for 9 days
blocked the isoprenaline infusion-induced increase in the per
centage of natural killer cells (Fig 1), but had no influence on
changes within the population of T-cells: Thelper/Tguppre3sor/cytolytic -cell ratio still decreased from 1.73
± 0.16 to 1.06 ± 0.09 (n=8, p=0.0007) following isoprenaline
infusion (Fig 2).
bisoprolol Celiprolol propranolol
Figure 2. The effect of xnfusion of isoprenaline on the ratio of helper to
suppressor/cytolytic T-cells of healthy volunteers under control conditions and after treatment with the ß-Ыоскег bisoprolol (n=8), Celiprolol (n=7)
and propranolol (n=8). For each group the first two columns represent
pre-treatment values before and after infusion of isoprenaline, respectively,
and the thirth and fourth column represent post-treatment values before and
after infusion of isoprenaline, respectively. Shown are means ± SEM of η subjects. ** P<0.01 vs. the corresponding pre-infusion value.
109
Effects of acute propranolol administration on isoprenaline
infusion-induced changes in lymphocyte subset composition.
Since a 9 day propranolol treatment had no effect on isoprenaline
infusion-induced changes in lymphocyte subset composition (and
decrease in the Thelper/Tsuppreggor/cytolytic -cell ratio) we studied
the effects of acute propranolol administration on these para
meters. For this purpose 5 healthy volunteers were administered
with 5 mg propranolol intravenously 45 minutes before the infusion
of isoprenaline. In this dose, propranolol markedly attenuated
the isoprenaline-induced increase in heart rate (data not shown),
indicating that the dose was sufficient to antagonize ß-adre-
noceptor mediated effects. However, as shown in Table 1, iso
prenaline still caused a granulocytosis and lymphocytosis. Within
the population of T-cells, lymphocytosis concerned
suppressor/cytolytic cells only. Since the absolute number of
helper T-cells did not change, the percentage of helper T-cells
decreased. The Thelper/T3uppressor/cytolytic -cell ratio accordingly
decreased from 1.44 ± 0.09 to 0.84 ± 0.02 (n=5, p=0.004). Fur
thermore pan-B and NK cell numbers increased following infusion
of isoprenaline.
Effects of ß-blocker treatments on lymphocyte in vitro pro
liferative responsiveness. 3H-thymidine uptake of lymphocytes upon stimulation with mitogenic
agents was taken as a measure of their ability to proliferate.
Compared to pre-treatment responses, no changes were found after
a 9 day ß-blocker treatment (Fig 3). After infusion of isopre
naline, in vitro proliferative responses of lymphocytes upon
stimulation with all five mitogens had decreased, in agreement
with recently published data from our laboratory (9). Treatment
with bisoprolol or Celiprolol for 9 days had no effect on this
isoprenaline infusion-induced depression of the proliferative
responses. However, a 9 day treatment with propranolol completely
prevented the isoprenaline infusion-induced depression of the
proliferative responses (Fig 3).
110
TABLE 1 : ISOPRENALINE INFUSION-INDUCED CHANGES IN CIRCULATING LEUKOCYTES IN
5 HEALTHY SUBJECTS ACUTELY PRETREATED INTRAVENOUSLY WITH PROPRANOLOL
Leukocytes
Granulocytes
Lymphocytes
Pan-T cells
Thelper cells
Tsuppressor/cytolytic
cells
Pan-B cells
Natural Killer cells
Thelper/
Tsuppressor/cy
tolytic cell ratio
Isoprenaline Infusion
Before
Rel (*)
71.0±2.4
40.2±1.6
28.2*1.1
16.4±1.4
2.0±0.5
1.44
Abs
(cells/μΐ)
4980±429
3140±304
1840±178
1314±155
745±90
519+58
304±41
35±8
tO.09
After
Rel (%)
71.8±3.2
31.0±0.8**
37.4±1.2**
22.4±2.0**
9.6±2.0*
Abs
(cells/μΐ)
6440±367*
3900+272
2560+223**
1864±239*
799±87
967±43**
578±82**
241±25*
0.84±0.02**
Relat ive p r o p o r t i o n s ( r e l %) of lymphocyte subsets were obtained as a percentage of t h e t o t a l lymphocytes counted (see Methods). The absolute (abs) number in each subset was c a l c u l a t e d by mult iply ing t h e percentage of each subset with the absolute lymphocyte count derived from t h e t o t a l leukocyte count and d i f f e r e n t i a l count . Values are means ± SEM. * P<0.05, ** P<0.01 vs. t h e corresponding pre- infus ion va lue.
DISCUSSION
During the l a s t decade, several authors have shown e f fec t s of
catecholamines (and other compounds which e levate i n t r a c e l l u l a r
c y c l i c AMP content) on c e l l u l a r immune functions in vitro (for
reviews see 1,3,14,15). Thus, increases in the i n t r a c e l l u l a r
leve l s of c y c l i c AMP i n h i b i t mitogen- or antigen-induced T-cel l
p r o l i f e r a t i o n , cytotoxic Τ lymphocyte function, lymphokine
s e c r e t i o n , and production and e f fect s of 11-2 (9,16-18).
Accordingly, in physiological and pathologica l condi t ions of
e levated plasma catecholamine l e v e l s , c e l l u l a r immune function
might be a l t e r e d . An impaired in vitro p r o l i f e r a t i v e respon
siveness a f t e r acute increases in endogenous catecholamines іл
111
2S0
200
150
100
M s o p r o l o l C e l i p r o l o l p roprano lo l
к РВА .
*чН Ν*
κ
E α. υ
140
100
во
20
100
во
20
ι н
Чн Ί
bofor· after boforo tftar before after
laoprenalloe Infuelon
Figure 3. In vitro proliferative responses of lymphocytes isolated before and
after infusion of isoprenaline in healthy volunteers under control conditions
(dashed lines) and after treatment (solid lines) with the ß-blockers bisoprolol (n=8), Celiprolol (n=7) and propranolol (n=B). Immediately before and after 25 min of infusion of isoprenaline heparinized blood was withdrawn and lymphocyte in vitro proliferative responses to PHA (1.8 μq/ml), Con A (9 pg/ml),
PWM (4.6 μg/ml), ATG (360 fjg/ml ) and STE (0.09 pg/ml) were determined as
described in Methods. Ordinates, [Эщ-thymidine incorporation into lymphocytes
in counts per min (cpm) χ 10~3. Values are means ± SEM of η experiments. *
P<0.05, ** P<0.01.
vivo, supplies indirect evidence for adrenergic control of immune
function (9,19). This depression of the immune responsiveness is
accompanied by a relative lymphocytosis with a predominant rise
in suppressor/cytolytic Τ cells and natural killer cells
(9,10,20). Moreover, recently Maisel et al (20) demonstrated a
112
reduced number of circulating lymphocytes in patients with chronic
heart failure, with a decrease in suppressor/cytolytic Τ and
natural killer cells but no change in helper Τ cells. This
alteration could be reproduced by treatment of healthy volunteers
with the lÎ2~adrener9:i-c agonist terbutaline. Whether these
alterations in lymphocyte numbers play a role in the depressed
immune responsiveness is not known.
In the present study, 9 day treatments with the ß-blockers
bisoprolol, Celiprolol or propranolol had neither effect on in
vitro proliferative responsiveness nor on circulating lymphocyte
numbers, indicating that the basal sympathetic tone does not play
an important role in control of immune function, at least via
ß-adrenergic receptors.
In agreement with the above mentioned findings, infusion of
isoprenaline caused changes in circulating lymphocyte numbers
and a depression of lymphocyte in vitro proliferative responses.
The depression of in vitro proliferation could be prevented by
a 9 day treatment with propranolol but not with bisoprolol or
Celiprolol, thus indicating involvement of the ß2-adrenoceptor
in the effect.
The depression of lymphocyte proliferative responsiveness seemed
to be not simply caused by the changes in the phenotypes of the
lymphocytes present in the circulation, since after propranolol
treatment lymphocyte numbers still changed following infusion
with isoprenaline but, in vitro proliferation was not depressed.
The reduced proliferative responses of the lymphocytes after
infusion with isoprenaline might be intrinsic to freshly released
lymphocytes. Freshly released lymphocytes have been suggested to
carry more ß2-adrenergic receptors than those found in the
circulation (9) . However, since no catecholamines are present
during the proliferation assay, these receptors might be of no
influence. Still, this is an indication that other more relevant
parameters, e.g. transmembrane signalling, function of protein
kinase С or the production, secretion and effects of lymphokines,
in freshly released lymphocytes can be different as well.
113
Accordingly, the release of these lymphocytes, expressing altered
intrinsic properties towards mitogenic stimulation, should be
ß2-adrenoceptor-mediated. Furthermore, since neither Celiprolol
or bisoprolol, nor propranolol, independently of to whether given
acutely or chronically, affect the distribution of lymphocytes
that express normal proliferative responses, it is hypothesized
that either distribution of these lymphocytes is not ß-adreno
ceptor mediated or an atypical ß-adrenoceptor is involved.
Recently, besides the classical ßj- and ß2-adrenoceptors (21),
an additional subtype of ß-adrenoceptors has been suggested to
mediate the sympathetic control of various metabolic processes
(22,23). Evidence for the existence of such atypical ß-adreno
ceptor sites includes their low affinity for standard ß-adre
noceptor blockers in binding as well as in functional experiments.
Moreover, recently a human gene has been isolated that encodes
an atypical ß-adrenoceptor, referred to as the '^-adrenergic
receptor" (22). The receptor protein was expressed in Chinese
hamster ovary (CHO) cells transfected with the cloned gene and
was found to posses atypical ß-adrenoceptor properties. For
example, besides all three catecholamines the atypical agonist
BRL 37344 also is a full agonist, and propranolol is a poor
inhibitor of ß-adrenoceptor mediated cyclic AMP accumulation in
СНО-Вз cells. The ß3-adrenoceptor is postulated to mediate
lipolytic effects of catecholamines. The present finding that
propranolol was ineffective in blocking the isoprenaline infusion
induced redistribution of circulating lymphocyte subsets, could
be an indication that the ß3-adrenergic receptor is involved.
However, more studies are necessary to proof this hypothesis.
In conclusion, ß-blocker treatment in healthy volunteers does
not affect lymphocyte function in basal conditions. This argues
against an important role of the sympathetic nervous system to
regulate immune function via ß-adrenoceptors. Thus, chronic
ß-blocker therapy might be expected to cause no important changes
in immune function. However, in states of short-term activation
of the sympathetic nervous system lymphocytes may be freshly
114
released into the circulation. Part of these lymphocytes have a
depressed proliferative capacity, probably due to altered
intrinsic properties, and are released via stimulation of
ß2-adrenoceptors. The other part of lymphocytes appear to express
normal proliferative capacity and are released either by a
ß-adrenoceptor independent pathway, or via stimulation of an
atypical ß-adrenoceptor. The physiological significance of this
differential release of lymphocytes is not known.
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14. Bourne HR, Lichtenstein LM, Melmon KL, Henney CS, Weinstein
Y, Shearer GM. Modulation of inflammation and immunity by cAMP:
receptors for vasoactive hormones and mediators of inflammation
regulate many leucocyte functions. Science 1974;184:19-29.
15. Hadden JW. Transmembrane signals in the activation of Τ
lymphocytes by mitogenic agents. Immunol Today 1988;9:235-9.
16. Feldman RD, Hunninghake GW, McArdle WL. ß-Adrenergic
receptor-mediated suppression of interleukin 2 receptors in human
lymphocytes. J Immunol 1987;139:3355-9.
17. Mary D, Aussei С, Ferrua В and Fehlman M. Regulation of
interleukin 2 synthesis by cAMP in human Τ cells. J Immunol
1987;139:1979-1984.
18. Johnson KW, Davis BH and Smith KA. CAMP antagonizes interleukin
2-promoted T-cell cycle progression at a discrete point in early
Gj,. Proc Natl Acad Sci USA 1988;85:6072-6076.
19. Crary B, Borysenko M, Sutherland DC, Kutz I, Borysenko JZ
116
and Benson H. Decrease in mitogen responsiveness of mononuclear
cells from peripheral blood after epinephrine administration in
humans. J Immunol 1983;130:694-697.
20. Maisel AS, Knowlton KU, Fowler P, Rearden A, Ziegler MG,
Motulsky HJ, Insel PA and Michel MC. Adrenergic control of
circulating lymphocyte subpopulations. Effects of congestive
heart failure, dynamic exercise, and terbutaline treatment. J
Clin Invest 1990;85:462-467.
21. Lands AM, Arnold A, McAuliff JP, Luduena FP and Brown TG.
Differentiation of receptor systems activated by sympathomimetic
amines. Nature Lond 1967;214:597-598.
22. Emorine LJ, Marullo S, Briend-Sutren M-M, Patey G, Tate K,
Delavier-Klutchko С and Strosberg AD. Molecular characterization
of the human ß3-adrenergic receptor. Science 1989;245:1118-1121.
23. Zaagsma J and Nahorski SR. Is the adipocyte ß-adrenoceptor
a prototype for the recently cloned atypical '^-adrenoceptor"?
Trends Pharmacol Sci 1990;11:3-7.
117
CHAPTER 6. Reduced proliferation of human lymphocytes after
infusion of isoprenaline is unrelated to impair
ments in transmembrane signalling
Abstract
Peripheral blood lymphocytes of normal healthy volunteers exposed
to acute stress or elevated concentrations of exogenously applied
adrenaline or isoprenaline, have a reduced proliferative capacity
in response to mitogenic stimulation with lectins. Although the
effect is accompanied by a redistribution of lymphocyte subsets,
the altered phenotypic distribution of T-cells does not explain
the reduced proliferative responses. We examined whether the
effect is due to a decreased intrinsic efficiency of transmembrane
signalling.
Isoprenaline was infused into normal healthy volunteers for 25
min at increasing doses. Lymphocytes obtained after the infusion
incorporated significantly less [3H]-thymidine when stimulated
with mitogenic lectins, and the proportions of different phe-
notypes of lymphocytes present in the circulation, had changed.
Thus, suppressor/cytolytic T-cells and natural killer cells had
increased, and helper T-cells had decreased.
Comparison of second messenger responses of lymphocytes obtained
before and after infusion of isoprenaline showed that upon
stimulation with PHA or Con A, lymphocytes obtained after infusion
of isoprenaline exhibited a higher formation of inositol phos
phates and a higher or at least equal [Ca',","]i response compared
with lymphocytes obtained before infusion of isoprenaline. Thus,
the reduced proliferative capacity of lymphocytes obtained after
infusion of isoprenaline cannot be attributed to impairments in
membrane-associated events in signal transduction.
Introduction
Immune homeostasis is the result of a delicate balance of subsets
of the immunocompetent lymphocytes and can be modulated by
endocrine and nervous systems (1-4), including the sympathetic
118
nervous system (for reviews see 5-7). Jn vitro, catecholamines
inhibit various functions of lymphocytes (5-10) . Also in vivo,
stress is increasingly reported in association with immunosup
pression (11-16). Acute exposure of normal healthy volunteers to
elevated concentrations of circulating adrenaline, either
increased by insulin-induced hypoglycemia (17) or applied
exogenously (18), causes a depression of the proliferative
capacity of circulating lymphocytes. The activation of resting
lymphocytes is a multistep process involving early biochemical
alterations such as formation of inositol phosphates and increases
in intracellular free calcium leading to activation of protein
kinase С (19-21). Administration of adrenaline or isoprenaline
to human subjects causes an absolute and relative lymphocytosis
(14,16,17). In addition, alterations occur in the relative
proportions of major lymphocyte subclasses present in the
circulation (22,23). Since intracellular free calcium responses
of subpopulations of peripheral blood lymphocytes are hetero
geneous (24) , these alterations in lymphocyte numbers could
contribute to the depression of the proliferative response towards
mitogenic lectins. However, we have recently shown that the
depression of proliferative capacity can be prevented despite
alterations in lymphocyte numbers (25). Apparently, the changes
in the phenotypical profile of the circulating lymphocytes may
be not responsible for the reduced proliferative responses.
Alternatively, the reduced mitogenic responsiveness of lympho
cytes after infusion with isoprenaline might be due to altered
intrinsic properties of the freshly released lymphocytes. For
example, freshly released lymphocytes have been shown to carry
more ß2-adrenergic receptors than those found in the circulation
(23). The aim of the present study was to determine whether an
altered efficiency of transmembrane signalling underlies the
depression of proliferative responses. For this purpose, before
and immediately after infusion of isoprenaline in young healthy
volunteers, blood was withdrawn by venipuncture and formation of
inositol phosphates and elevations of intracellular free Ca++ in
lymphocytes upon stimulation with the mitogenic lectins phy-
119
tohemagglutinin and concanavalin A, were determined. In addition,
circulating lymphocyte subsets were analyzed and incorporation
of [3H]-thymidine after the lectin challenge was assessed.
Materials and Methods
Experimental protocol Six young healthy volunteers (aged 25-29 yr) participated in the
study after having given informed written consent. At 0800 h an
indwelling cannula was inserted into a forearm vein. In the
opposite forearm a needle was placed into a large vein and kept
patent with infusion of 0.9 % saline. After one hour of rest in
the supine position, isoprenaline was infused in sequential doses
of 3.5, 7, 17.5, 35 and 70 ng·kg-1 -min"1 for 5 min each. Before
and immediately after the infusion, blood samples were withdrawn
after first flushing saline from the infusion needle with about
3 ml blood. For analysis of lymphocyte subsets and for measurements
of second messenger responses upon mitogenic stimulation, blood
was anticoagulated with EDTA, for assessment of lymphocyte
proliferative responses blood was anticoagulated with heparin
(500 IU heparin/10 ml blood).
Cell isolation Peripheral blood lymphocytes were prepared from whole blood by
Ficoll gradient centrifugation according to the method of Böyum
(26) with minor modifications (23).
Analysis of lymphocyte subsets Lymphocyte subsets were analyzed by indirect immunofluorescence
with the following monoclonal antibodies: Jot3 for pan-T cells;
Jot4a and BMA040 for helper T-cells; Jot8a and BMA080 for sup-
pressor/cytolytic T-cells; aDR for pan-B cells; JotlO for natural
killer cells. LeuMl was used for quantification of monocytes and
granulocytes present in the cell suspensions. Fluorescein iso-
thiocyanate (FITC)- labeled goat anti-mouse immunoglobulin G
(IgG) + IgM was used as the second antibody. In short, lymphocytes
were incubated with appropriate dilutions of monoclonal antibody
for 40 min, washed, labeled with FITC-conjugated goat antimouse
120
antibody for 40 min and washed; all procedures were performed at
room temperature. Cells were analyzed within 2 h under a Zeiss
fluorescence microscope (Zeiss, Oberkochen, Germany).
3H-Thymidine incorporation Lymphocytes were stimulated to proliferate with the mitogenic
lectins phytohemagglutinin (PHA; 0.45, 0.9, 1.8 and 3.6 ßg/ml)
and concanavalin A (Con A; 4.5, 9, 18 and 36 дд/ml). The cells
were cultured for 4 days at 37 "C in microtiter plates containing
the lectins in 220 μΐ RPMI-1640 with 0.29 mg/ml L-glutamine, 0.16
mg/ml gentamycin-sulfate, and 10% heat-inactivated pooled human
serum at 2·105 cells/well. For the last 16 h the cultures were
labeled with 2 дСі [3H]-thymidine/well. The cells were harvested
with a multiple sample precipitator (Skatron, Lier, Norway), and
incorporated radioactivity was assessed in a liquid scintillation
counter (LKB 1212, Freiburg, Germany).
Second messenger responses
Formation of inositol phosphates (IPs) was determined according
to previously published techniques (27) . In short, lymphocytes
were labeled with [3H]-myoinositol in Krebs-Henseleit solution
for 2 h at 37 "С, washed, stimulated and incubated for another
30 min at 37 "C in the presence of 10 mM LiCl. PHA (1.8 ßg/ml)
and Con A (18 ßg/ml) were used as stimuli. Accumulated
water-soluble [3H]-IPs were extracted with a water-metha-
nol/chloroform system and loaded onto Dowex anion exchange resin
columns. [3H]-IPs were eluted in one fraction and counted in a
Beekman LS 9000 liquid scintillation counter (Beekman Inst.,
Fullerton, CA) at an efficiency of about 42%.
Intracellular free calcium ([Ca++]i) was measured with the flu
orescent dye Fura-2 as previously described (27) . For this purpose,
lymphocytes were incubated with 1 μΜ Fura-2 for 1 h at room
temperature, washed and stimulated with various concentrations
of PHA or Con A. For conversion of raw fluorescence data into
[Ca++]i, the presence of extracellular Fura-2 was taken into
account.
121
Data evaluation
All data are shown as means ± SEM of η experiments. Differences
between groups were compared with two-tailed paired t-test; a
p-value smaller than 0.05 was considered to be significant.
Materials
[3H]-thymidine (5 Ci/mmol) was purchased from Amersham and
Myo-[2-3H(N)]-inositol (specific activity 20 Ci/mmol) was from
New England Nuclear (Dreiech, FRG).
Isoprenaline (Aludrin) was obtained from Boehringer (Ingelheim,
FRG) .
FITC coupled goat anti-mouse IgG + IgM was purchased from Medac
(Hamburg, FRG). Monoclonal antibodies were obtained from the
following sources (specificities in parenthesis): Jot3 (CD3),
Jot4a (CD4), JotSa (CD8) and JotlO (natural killer cell marker)
from Immunotech Dianova (Hamburg, FRG); LeuMl (pan-monocy-
te/granulocyte marker) and aDR (class II) from Becton Dickinson
(Heidelberg, FRG); BMA040 (CD4) and BMA080 (CD8) from Behring
(Frankfurt/Main, FRG).
RPMI 1640, L-glutamine and gentamycin-sulfate were obtained from
Nunc (Wiesbaden, FRG). PHA was from Welcome (Beckenham, U.K.),
Con A from Sigma (Deisenhofen, FRG).
Dowex l-x8 anion exchange resin (200-400 mesh, formate form) was
obtained from Bio-Rad Laboratories (Richmond, USA), Fura-2
acetoxymethoxyester was obtained from Molecular Probes (Eugene,
OR) .
All other chemicals were from Sigma and of the highest purity
grade available.
Results
Effects of isoprenaline infusion on lymphocyte subset composition
The number of granulocytes and monocytes present in the lymphocyte
suspensions did not exceed 5% and was similar for all preparations.
Infusion of isoprenaline did not change the relative amounts of
pan-T cells and pan-B cells (Table 1) . However, within the
122
population of T-cells, the percentage of helper cells decreased
(38.2 ± 1.7 before vs. 30.8 ± 1.9 after infusion, n=6, p=0.09)
and of suppressor/cytolytic cells increased (24.3 ± 2.0 before
vs. 33.0 ± 2.4 after infusion, n=6, p=0.007). Accordingly,
Theiper/Tsuppressor/cytoiytic -cell ratio, a commonly used indicator
of immunoregulatory cell disbalance (28), decreased from 1.62 ±
0.12 to 0.98 + 0.14 (n=6, p=0.02). The percentage of natural
killer cells increased from 5.8 ± 1.7 to 9.0 ± 1.8 (n=6, p=0.15)
following isoprenaline infusion.
TABLE 1: RELATIVE DISTRIBUTION OF LYMPHOCYTE SUBSETS IN 6 HEALTHY SUBJECTS
BEFORE AND AFTER INFUSION OF ISOPRENALINE
PAN-T CELLS
HELPER-T CELLS
SUPPRESSOR/CYTOLYTIC-T CELLS
PAN-B CELLS
NATURAL KILLER CELLS
HELPER TO SUPPRESSOR/CYTOLYTIC CELL
RATIO
BEFORE
67.512.6
38.2±1.7
24.3±2.0
16.812.9
5.811.7
1.6210.12
AFTER
70.312.4
30.811.9
33.012.4*
20.511.6
9.011.8
0.9810.14*
Immediately before infusion and after 25 min of infusion of isoprenaline,
blood was withdrawn for determination of distribution of lymphocyte subsets.
Values are means 1 SEM of 6 experiments. * Ρ < 0.05 vs. the corresponding
preinfusion level.
Lymphocyte proliferative responses
After a 4 day culture in the presence of the mitogenic lectin
PHA or Con A, lymphocytes obtained before infusion of isoprenaline
incorporated significantly more [3H]-thymidine than did lym
phocytes obtained immediately after infusion of isoprenaline
(Figure 1).
Forjnation of [3H]-IPs
Incorporation of [3H]-myoinositol was identical in lymphocytes
obtained before and after infusion of isoprenaline (data not
shown) . Basal formation of [3H]-IPs in lymphocytes obtained before
or after infusion of isoprenaline was similar (Figure 2) . However,
123
M
о 2 0 0
χ 150
& loo
50
л
η=5
• fr-H-î • i-i-ï-i
ï * · * •
1 IPHA]. ug/nl
10 10 100 (Con A), pg/mi
Figure 1. Lectin-stimulated incorporation of (^Н)-thymidine in human lym
phocytes obtained before (open circles) and after (closed circles) infusion
of isoprenaline. Immediately before infusion and after 25 min of infusion
(see Materials and Methods: experimental protocol), hepannized blood was
withdrawn and lymphocyte in vitro proliferative responses to PHA and Con A
were determined as described in Waterials and Methods. Ordinates, (^HJ-thy-
midine incorporation into lymphocytes in counts per min (cpm) χ lO-^; abscissae,
lectin concentrations in pg/ml. Values are means ± SEM of η experiments. * Ρ
< 0.05 vs. the corresponding preinfusion level.
lectin-stimulated formation of IPs was significantly greater in
lymphocytes obtained after infusion of isoprenaline. Upon
stimulation with 1.8 ßg/ml PHA the response was about 5 times as
large, and upon stimulation with 18 ßg/ml Con A the response was
about 2.5 times as large in lymphocytes obtained after infusion
of isoprenaline compared to lymphocytes obtained before infusion
of isoprenaline (Figure 2).
[Ca++]i measurements
Basal levels of [Ca++]i of lymphocytes obtained before and after
infusion of isoprenaline were similar (60.2 ± 2.5 nM before vs
64.4 ± 2.1 nM after infusion of isoprenaline, n=5). PHA and Con
A concentration-dependently elevated [Ca++]i to a peak level
within 1-3 min. From the peak [Ca++]i elicited by 4-5 lectin
concentrations, we constructed concentration-response curves
(Figure 3). The curves for Con Α-stimulated increases in [Ca++]i
in lymphocytes obtained before and after infusion of isoprenaline
were superimposable. PHA-stimulated increases in [Ca++]i were
124
_ 2500
S 2000
<n 1500 Q_
¿ 1000 «о
500
blank nu - blank Con I - blank
Figure 2. Formation of [^HJ-inositol phosphates (IPs) in human lymphocytes
obtained before (open columns) and after (hatched columns) infusion of iso-
prenaline. Lymphocytes, prelabeled with [ HJ-myoinositol, were incubated for
30 min in the presence and absence of PHA (1.8 μg/ml) or Con A (18 pg/ml) at
370C and formation of [^HJ-IPS was determined as described in Materials and
Methods. Ordinate, formation of [^HJ-IPs m counts per min (cpm) per 5 10°
lymphocytes. Values are means ± SEM of б experiments. * Ρ < 0.05 vs. the
corresponding preinfusion level.
slightly but significantly higher in lymphocytes obtained after
infusion of isoprenaline compared to lymphocytes obtained before
infusion of isoprenaline.
s с
tu in га αϊ С-(J
с f-i
г co о
100
75
50
25
0
* ' I
. Ρ ,
1 10 1 ΙΡΗΑ1, /»g/ni
00
100
75
50
25
0 10 100 1000 [Con A], íi g/ml
Figure 3. Lectin-stimulated increases of [Ca++]i in human lymphocytes obtained before (open circles) and after (closed circles) infusion of isoprenaline. Lymphocytes were incubated with various concentrations of PHA and Con A at 250C and alterations of (Ca++)i were monitored with Fura-2. Ordinate, increase of (Ca++]i in nM; abscissae, lectin concentrations in pg/ml. Values are means ± SEM of 5 experiments. * Ρ < 0.05 vs. the corresponding preinfusion level.
125
Discussion
The human immune system consists of discrete subsets of immu
nocompetent lymphocytes that are critical for immune homeostasis
(28). The present study shows that intravenous administration of
isoprenaline to human subjects produces a shift in the proportion
of certain lymphocyte subsets in the peripheral blood. Although
the percentage of pan-T cells remained unchanged following
infusion of isoprenaline, the relative amount of helper cells
decreased and that of suppressor/cytolytic cells increased.
Previous studies showed that the decline in the percentage of
helper T-cells is due to both an increase in the absolute numbers
of natural killer cells and suppressor/cytolytic lymphocytes, as
well as a decrease in the absolute number of helper T-cells (23).
A disturbed lymphocyte migration into peripheral tissues (29,30)
as well as an altered adherence of lymphocytes to endothelial
cells and tissue-specific receptors (31-33) might account for
the change in lymphocyte numbers. Isoprenaline may act directly
by activation of lymphocyte ß2-adrenergic receptors (23,34), or
indirectly, by activation of ß-adrenergic receptors on other
tissues.
The outcome of an immune response largely depends on the number
of lymphocytes and monocytes, as well as on the composition of
lymphocyte subsets. Human T-lymphocytes are endowed with the
capacity to recognize specific antigens, execute effector
functions, and regulate the type and intensity of virtually all
cellular and humoral immune responses. Perturbations in subset
dynamics may initiate a variety of immunopathologic disorders
(28).
In the present study, lymphocytes obtained after infusion of
isoprenaline showed a reduced proliferative capacity in response
to the mitogenic lectins PHA and Con A. Previous studies indicated
that the alterations in T-lymphocyte subsetcomposition may be
not responsible for the reduced proliferative responses: infusion
of isoprenaline in healthy volunteers treated with propranolol
induced an increase in the percentage of suppressor/cytolytic
126
cells and a decrease in the percentage of helper cells but did
not affect lymphocyte іл vitro proliferative responses to various
mitogenic agents (25). It was hypothesized that the effect might
be due to intrinsic properties of freshly released lymphocytes.
In the present study we investigated the efficiency of transduction
of the signal across the membrane (19-21). Our results suggest
that the reduced proliferative capacity of lymphocytes obtained
after infusion of isoprenaline is not related to decreased
efficiency of transmembrane signal transduction. Lymphocyte
activation involves very early increases in [Ca',",']
i and formation
of IPs. A decreased production of IPs and/or an attenuated increase
in [Ca+ +]
i could result in a reduced proliferative respons.
However, after infusion of isoprenaline PHA-stimulated formation
of IPs was markedly, and [Ca++]i responses were slightly increased;
Con Α-stimulated formation of IPs had increased somewhat less
and [Ca++]i responses were similar. According to these findings,
proliferative capacity might be expected to increase or at least
remain constant, but in fact it was decreased (see above). The
enhancement of second messenger responses might be caused by a
higher number and/or by an increased affinity of the receptors
for the lectins in lymphocytes obtained after infusion of iso
prenaline. Freshly released lymphocytes have recently been
reported to carry about twice as many ß2-adrenergic receptors
than those already present in the circulation (23).
Alternatively, proliferative abnormalities might be caused by
alterations in activation steps subsequent to transmembrane
signalling, for example the activation and/or function of protein
kinase С (35,36), or the production, secretion and effects of
lymphokines (4,37,38).
Observations regarding in vitro treatment of lymphocytes with
catecholamines have been rather conflicting. Crary et al. (18)
reported that incubation of human mononuclear cells with adre
naline had no effect on the incorporation of [3H]-thymidine
following mitogenic stimulation with PHA or pokeweed mitogen. On
the other hand, inhibitory effects of catecholamines on mitogen-
127
or antigen-induced T-cell proliferation, cytotoxic T-lymphocyte
function, lymphokine secretion, and production and effects of
IL-2 have been reported (5-10). Moreover, we have recently shown
inhibitory effects of catecholamines on lectin stimulated for
mation of IPs and elevation of [Ca++]i in human lymphocytes in
vitro (27) . However, since 1> the in vitro effects are found in
the presence of the catecholamine only, and 2) ex vivo increases
in transmembrane signalling are found (this study), different
mechanisms seem to underlie the effects of catecholamines in vivo
and іл vitro. It is suggested that in vivo the catecholamine acts
indirectly, causing an exchange of lymphocytes between the
circulation and peripheral tissues, whereby the freshly released
lymphocytes might have altered intrinsic properties concerning
activation steps post transmembrane signal transduction.
We conclude that the reduced proliferative capacity towards
mitogenic lectins in lymphocytes obtained after infusion of
isoprenaline appears not to be due to a decreased efficiency of
transmembrane signal transduction. Future studies must show
whether the reduction in proliferative response of lymphocytes
in relation to acute increases in catecholamines, might be caused
by impaired activation pathways subsequent to transmembrane
signalling.
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SUMMARY
Circulating blood lymphocytes contain a homogeneous population
of ß2-adrenoceptors. These cells are frequently used to monitor
changes of ß-adrenoceptor function in humans. This thesis presents
in vitro and іл vivo studies which were designed to investigate 1) the mechanisms underlying the dynamic regulation of lymphocyte
ß2-adrenoceptors and 2> the role of lymphocyte ß2-adrenoceptors
for human immune function.
Jn vitro, ßj-adrenoceptors in lymphocytes are subject to
homologous and heterologous desensitization in a manner similar
to that observed in various other model systems. Catecholamines
induced a time- and concentration-dependent homologous
desensitization of ßj-adrenoceptor function with an order of
potency typical for a ß2-adrenoceptor: isoprenaline > adrenaline
>> noradrenaline. The phorbol ester 12-o-tetradecanoyl-
phorbol-13-acetate (TPA) was shown to cause heterologous
desensitization of the lymphocyte ß2-adrenoceptor, possibly by
an impairment of the function of the Gs-protein and/or the ability
of the ß-adrenoceptor to couple to GB.
Jn vivo, acute exposure to catecholaminergic agonists caused
an increase of ß2-adrenoceptors on circulating blood lymphocytes,
in contrast to what has been observed in all other model systems.
Measurements of intracellular cAMP content and of adenylate
cyclase activity showed an increased isoprenaline-induced cAMP
generation after infusion of isoprenaline, suggesting that the
additional receptors were functional. The effect was ß2-adre-
noceptor mediated, since (1) adrenaline was much more potent than
noradrenaline and (2) it could be abolished by acute pretreatment
of the volunteers with the ß2-selective antagonist ICI 118,551
but not with the ßj^-selective antagonist bisoprolol.
It seemed impossible to induce similar increases in ß2-adre-
noceptor density in vitro. Incubation of lymphocytes, obtained
before infusion of isoprenaline, in autologous plasma obtained
immediately after the infusion did not influence ß2-adrenoceptor
132
density at all. Thus, unless a local and short-living substance
is involved, the possibility that the alteration was caused by
direct effect of substances released in the plasma during the
infusion (for example adrenal corticosteroids), was unlikely but
can not be ruled out completely. Furthermore the phenomenon was
lymphocyte specific (no alteration was observed in platelet or
monocyte ß2-adrenoceptors).
Lymphocytes are not a homogeneous population but consist of
different subsets and, acute exposure to catecholamines іл vivo
is known to cause a lymphocytosis. Analysis of the subsetcom-
position of circulating blood lymphocytes following infusion of
isoprenaline revealed unequal changes in lymphocyte numbers.
Whereas the number of pan-B cells and pan-T cells remained fairly
constant, the number of NK cells increased. Among the T-cells T3uppressor/cytolytic cells increased and T
h e l p e r cells decreased.
Infusion of noradrenaline, which did not affect ß2-adrenoceptors,
did not change lymphocyte numbers.
In case of a different distribution of ß2-adrenoceptors on
lymphocyte subsets, this alteration of the subsetcomposition of
circulating blood lymphocytes might contribute to the increase
in ß2-adrenoceptors on unfractionated lymphocytes following
infusion of isoprenaline. Indeed, there were marked differences
in ß2-adrenoceptor densities of different lymphocyte subsets.
Pan-B cells had significantly more ß2-adrenoceptors than pan-T
cells, and among the T-cells, Tsuppre3sor/cytolytic cells had more
ß2-adrenoceptors than Thelper cells. These differences however,
were not large enough to explain the 2-fold increase in
ß2-adrenoceptor density on unfractionated lymphocytes. In fact,
the contribution of the alteration of the subsetcomposition to
the increase in ß2-adrenoceptors, seemed relatively unimportant,
since ß2-adrenoceptor density had increased in each subset.
The rapid time course of the increase of ß2-adrenoceptors on
circulating blood lymphocytes (within 15 minutes) makes de novo
synthesis of ß2-adrenoceptors very unlikely. It has been
postulated that sympathetic stimulation might shift receptors
133
from sequestered compartments to the cell surface. However,
increases in ß2-adrenoceptor density determined on intact cells
and on membranes were similar, and experiments to induce increases
in ß2-adrenoceptor density in vitro were not successfull. Thus
the rapid increase in lymphocyte ß2-adrenoceptors is not likely
to be caused by receptor regulation. The fact that receptors had
still increased on each subset (even on pan-B cells, which did
not change in number!) can be explained by assuming that an
exchange of lymphocytes had taken place, or in other words, that
the cells pre-infusion and the cells post-infusion were from
different populations. This exchange can not be determined
directly by the method available. However, the role of the exchange
process can be assessed by taking away an organ that might be
involved in it. The spleen is a large source of lymphocytes in
the body. The role of this organ was assessed in some patients
who had to undergo splenectomy. Infusion of isoprenaline in two
Hodgkin's patients before splenectomy induced similar changes in
ßj-adrenoceptor density and in lymphocyte subsetcomposition as
in healthy subjects. Thus, interference of the disease itself
could be excluded. After splenectomy, however, isoprenaline
infusion increased ß2-adrenoceptor density on unfractionated
lymphocytes only by 40%, and, apart from an increase in the
percentage of NK cells, lymphocyte subset composition was not
affected. The still remaining slight increase in ß-adrenoceptor
density in splenectomized patients could partly be explained by
the increase in the percentage of NK cells, which contain a rather
high number of ß-adrenoceptors. However, exchange of lymphocytes
between the circulation and secondary lymphoid organs other than
the spleen, like for example the thoracic duct, the lung or the
lymph nodes, is a more likely explanation.
Taken together, the rapid increase in ß2-adrenoceptors on
unfractionated lymphocytes following isoprenaline infusion does
not represent receptor regulation, but instead is a lymphocyte
specific phenomenon. It is possibly caused by a release of
lymphocytes into the circulation and/or by an exchange of lym
phocytes between peripheral lymphoid tissues and the circulation.
134
Hereby freshly released lymphocytes appear to carry more
ß2-adrenoceptors than those found in the circulation. The two
phenomena, however, are not strictly coupled. The pharmacological
characteristics of the isoprenaline infusion-induced redis
tribution of lymphocytes differed from those of the isoprenaline
infusion-induced increase in lymphocyte ß2-adrenoceptors. Whereas
the increase in ßj-adrenoceptors was ß2-adrenoceptor-mediated
(see above: inhibition by ICI 118,551 but not by bisoprolol),
neither bisoprolol or Celiprolol (administered chronically) nor
propranolol (independent whether administered acutely or
chronically) inhibited the isoprenaline infusion-induced
redistribution of lymphocytes. Apparently the exchange of lym
phocytes concerns "normal" as well as "receptor rich" lymphocytes.
Whereas the release of "receptor-rich" lymphocytes is
ß2-adrenoceptor-mediated, the release of "normal" lymphocytes is
neither ß ^ nor ß2-adrenoceptor-mediated.
In order to determine the relevance of alterations in lymphocyte
numbers and ß2-adrenoceptors for immune function, we assessed
the in vitro proliferative response of non-fractionated lym
phocytes towards mitogenic lectins. After isoprenaline but not
noradrenaline infusion, proliferative responsiveness was reduced.
Similar to the increases in ß2-adrenoceptors, this depression of
in vitro proliferation could be prevented by a 9 day treatment
with propranolol but not with bisoprolol or Celiprolol, thus
indicating involvement of the ß2-adrenoceptor in the effect.
Furthermore, since after propranolol treatment lymphocyte sub-
setcomposition still changed following infusion with isoprenaline
(see above) but, in vitro proliferation was not depressed, it
could also be concluded that the depression of lymphocyte pro
liferative responsiveness was not caused by the alteration in
lymphocyte subsetcomposition. These findings suggest a
relationship between ß2-adrenoceptor density and proliferative
responsiveness of circulating lymphocytes.
135
The reduced mitogenic responsiveness of the lymphocytes after
infusion with isoprenaline might be caused by altered intrinsic
properties of freshly released lymphocytes. An obvious candidate
for a role in this phenomenon is the ß2-adrenoceptor. Indeed, in
vitro stimulation of lymphocyte ß2-adrenoceptors inhibited the
lectin-induced accumulation of inositol phosphates and influx of
extracellular calcium, both very early events following lymphocyte
activation. This inhibitory effect seemed to be mediated by cAMP
since it was mimicked by prostaglandin E1 and forskolin and
enhanced by inhibition of phosphodiesterases. However, studies
on transmembrane signalling of lymphocytes obtained before and
after infusion of isoprenaline revealed that formation of inositol
phosphates and increases in intracellular calcium following the
lectin challenge were enhanced instead of decreased in lymphocytes
obtained after the infusion. Thus, although the inhibitory effect
of catecholamines on transmembrane signalling might take place
in vivo, it does not seem to play an important role in determining
lymphocyte in vitro proliferative capacity.
The reduced proliferative capacity of lymphocytes obtained
after infusion of isoprenaline, might be due to altered properties
concerning activation steps subsequent to transmembrane sig
nalling, like for example the activation and/or function of
protein kinase C, or the production, secretion and effects of
lymphokines.
The physiological significance of these catecholamine-induced
changes in lymphocyte immune function was investigated in the
model of insulin-induced hypoglycemia in healthy volunteers. The
nadir of decreased blood glucose levels following intravenous
injection of insulin was associated with an about 10-fold increase
in plasma adrenaline, but an only modest increase in plasma
noradrenaline. This vigorous increase in plasma adrenaline was
accompanied by an increase in functional lymphocyte ßj-adreno-
ceptors comparable with those evoked by infusion of exogenous
adrenaline or isoprenaline. Concomitantly, lymphocyte
subsetcomposition was markedly altered and proliferative
136
responses to various mitogenic lectins were attenuated. Thus,
acute changes in ß2-adrenoceptor function, subsetcomposition and
proliferative responses of peripheral blood lymphocytes do not
only occur in response to exogenously applied ß-adrenoceptor
agonists, but also in response to acute (patho)physiological
elevations of endogenous catecholamines. The effect was transient
and paralleled the changes in plasma catecholamines: when plasma
catecholamine levels had reached pre-drug levels, also the above
mentioned parameters had returned to the corresponding values
obtained before insulin application. Whether these transient
changes of circulating lymphocytes, accompanying acute changes
in plasma catecholamines, are a reflection of immune respon
siveness in vivo, remains to be elucidated.
Accordingly, in physiological and pathological conditions of
acutely elevated sympathetic nervous activity, (cellular) immune
function might be altered. The basal sympathetic tone, however,
does not seem to play an important role in control of immune
function via ß-adrenoceptors. Treatment with ß-adrenoceptor
antagonists (bisoprolol or Celiprolol or propranolol) for 9 days
did not influence circulating lymphocyte numbers and also no
changes were found in in vitro proliferative responses.
In conclusion, in vitro studies yielded indications for an
inhibitory role of lymphocyte ß2-adrenoceptors in immune function.
The in vivo situation, however, appeared to be much more complex.
Jn vivo basal sympathetic tone did not seem to play an important
role in control of immune function via ß-adrenoceptors, but acute
stimulation of the sympathetic nervous system (endogenously as
well as exogenously) affected two parameters of lymphocyte immune
function: 1) the subset composition of circulating lymphocytes
changed, probably due to an exchange of lymphocytes with secondary
lymphoid organs, and 2> the proliferative capacity of circulating
lymphocytes was reduced. Since freshly released lymphocytes appear
to carry more ß2-adrenoceptors than those already present in the
circulation, these lymphocytes could be expected to be more
137
sensitive to inhibitory effects of catecholamines. However, the
opposite was the case: transmembrane signalling had increased in
these lymphocytes.
Thus, the mechanisms regulating number and subset composition
as well as proliferative capacity of human circulating lymphocytes
remain to be elucidated.
138
SAMENVATTING
Prikkels die via sympathische zenuwbanen worden voortgeleid
resulteren uiteindelijk in de afgifte van de neurotransmitterstof
noradrenaline. Deze neurotransmitter overbrugt de afstand van
zenuwuiteinde tot cellen van het doelorgaan. Deze cellen bevatten
eiwit structuren, receptoren genaamd, waarop zeer specifiek de
neurotransmitter kan binden. De interactie van de neurotransmitter
met de receptor initieert een reeks van biochemische gebeurte
nissen in de cel welke uiteindelijk leiden tot een biologisch
effect. De receptoren van het sympathische zenuwstelsel worden
adrenoceptoren genoemd. Naar gelang de voorkeursvolgorde van de
catecholamines isoprenaline, adrenaline en noradrenaline om de
receptoren te bezetten, worden α- en ß-adrenoceptoren onder
scheiden. Een verdere onderverdeling in subtypen van α- en
ß-adrenoceptoren berust op verschillen in voorkeursvolgorden van
synthetische stoffen die de receptoren bezetten.
Lymfocyten bevatten een homogene populatie ß-adrenoceptoren
van het subtype ß2· Omdat lymfocyten vrij eenvoudig te isoleren
zijn uit perifeer bloed werden en worden deze cellen vaak gebruikt
om de functie van adrenerge receptoren te bestuderen en te meten,
soms in de hoop hiermee een maat te hebben voor de functie van
ß2-adrenoceptoren op andere weefsels. Het in dit proefschrift
beschreven onderzoek werd opgezet om de regulatie en de functie
van deze ßj-adrenoceptoren op humane lymfocyten te bestuderen.
De gevoeligheid van een cel voor een neurotransmitter is onder
andere afhankelijk van het aantal receptoren voor de neuro
transmitter op deze cel. Deze receptor dichtheid is een dynamische
grootheid die door een groot aantal fysiologische en
pathofysiologische variabelen gereguleerd wordt. Een algemeen
voorkomend verschijnsel is de afnemende gevoeligheid voor een
neurotransmitter onder invloed van langdurige stimulatie door
diezelfde of door een andere neurotransmitter. We spreken dan
van respectievelijk homologe en heterologe desensitisatie. In
deze studie wordt aangetoond dat de ß2-adrenoceptor op lymfocyten
zowel homologe als heterologe desensitisatie kan ondergaan. De
139
catecholamines isoprenaline, adrenaline en noradrenaline ver
oorzaakten een tijds- en concentratie-afhankelijke homologe
desensitisatie van de ß2-adrenoceptor functie op lymfocyten. De
volgorde van potentie was typisch voor een ß2-adrenoceptor:
isoprenaline > adrenaline >> noradrenaline. Aan homologe
desensitisatie ligt een afname van het aantal functionele
receptoren ten grondslag, dit door een verstoorde koppeling met
het G-eiwit eventueel gevolgd door internalisatie van de receptor.
De phorbol ester TPA induceerde heterologe desensitisatie van de
ß2-adrenoceptor op lymfocyten. Via welke weg
12-o-tetradecanoyl-phorbol-13-acetate (TPA) de functie van de
ß2-adrenoceptor op lymfocyten beïnvloedt, is niet duidelijk.
In tegenstelling tot de effecten van catecholamines op de
ß2-adrenoceptor van lymfocyten in vitro, veroorzaakte intrave
neuze toediening van isoprenaline of adrenaline, maar niet van
gelijke doses noradrenaline, aan een proefpersoon, een
dosisafhankelijke toename in functionele ß2-adrenoceptoren op
perifere lymfocyten. Dergelijke veranderingen werden niet
gevonden voor andere bloedcellen zoals bloedplaatjes of monocyten.
Omdat lymfocyten uit verschillende subpopulaties bestaan die
onderling zouden kunnen verschillen in receptor dichtheid is de
centrale vraag of veranderingen in receptor aantallen onder
invloed van isoprenaline of adrenaline veroorzaakt worden door
een reeële toename van receptoren op circulerende cellen of door
een verschuiving in de samenstelling van de lymfocyten populatie.
Een methode werd ontwikkeld om de verschillende subpopulaties te
scheiden, zodat metingen konden worden uitgevoerd aan gezuiverde
subpopulaties. De verschillende subpopulaties verschilden
onderling in ß2-adrenoceptor dichtheid. Verder bleek echter dat
de toename in receptor dichtheid onder invloed van intraveneuze
toediening van isoprenaline het resultaat is van een verhoogde
receptor dichtheid op elke subpopulatie.
De veranderingen in de receptor dichtheid gingen gepaard met
veranderingen in de samenstelling van de lymfocyten populatie.
Zo daalde bijvoorbeeld de ratio van helper en suppressor/cy-
140
tolytische T-cellen van een normaal waarde van ± 1.7 naar een
duidelijk verlaagde waarde van ±0.7. Aldus ontstond de hypothese
dat een uitwisseling van lymfocyten tussen secundaire lymfoïde
weefsels, zoals bijvoorbeeld de milt, en het perife bloed, waarbij
de "nieuwe" lymfocyten een hogere ß2-adrenoceptor dichtheid
bezitten, ten grondslag ligt aan bovengenoemde veranderingen in
ß2-adrenoceptor dichtheid. De bevinding dat zowel veranderingen
in de samenstelling van de lymfocyten populatie als ook veran
deringen in de receptor dichtheid minder duidelijk waren bij
patiënten waarbij de milt operatief was verwijderd, leverde een
indirecte aanwijzing voor de juistheid van deze hypothese.
Een indicatie omtrent de immuun functie van lymfocyten is de
capaciteit van lymfocyten om zich te vermeerderen (prolifereren).
Hiertoe worden de lymfocyten geactiveerd met mitogene agentia
zoals bijvoorbeeld de lectinen phytohemagglutinine en concana-
valin A. De activatie van rustende lymfocyten berust op een
cascade van gebeurtenissen. Aan de basis hiervan staan produktie
van inositol fosfaten en een stijging van de intracellulaire
calcium concentratie. Na intraveneuze toediening van isoprenaline
was de proliferatie van de perifere lymfocyten verminderd. Deze
verlaagde proliferatie capaciteit van de lymfocyten zou in verband
kunnen staan met de verhoogde ß2-adrenoceptor dichtheid op de
lymfocyten. Γη vitro stimulatie van de ß2-adrenoceptor remde de
produktie van inositol fosfaten en de stijging van de intra
cellulaire calcium concentratie tijdens de activatie van lym
focyten met mitogene agentia. De concentratie van adrenaline
waarbij deze werking optrad ligt in de nano-molaire range. Het
effect zou dan ook van (patho)fysiologische betekenis kunnen
zijn. Een remmende werking op deze vroege signalen tijdens de
activatie van lymfocyten zou de oorzaak kunnen zijn van een
verminderde proliferatie. Echter, analyse van deze vroege bio
chemische signalen aan lymfocyten welke werden geïsoleerd vóór
en ná intraveneuze toediening van isoprenaline, bracht niet een
verminderde maar eerder een sterk verhoogde signaal transductie
aan het licht.
141
Naast de toename van ß-agonisten door intraveneuze toediening
gaf ook een endogene stijging van plasma catecholamines effecten
op de perifere lymfocyten populatie. Intraveneuze toediening van
insuline aan gezonde vrijwilligers leidde tot een daling van de
glucose concentratie in het bloed. Deze hypoglykemie veroorzaakte
een stijging in de plasma adrenaline concentratie tot ongeveer
tien maal haar basale waarde. De plasma noradrenaline concentratie
daarentegen steeg nauwelijks. Parallel aan deze endogene toename
in plasma catecholamines veranderden ook de samenstelling van de
lymfocyten populatie, en de ß2-adrenoceptor dichtheid en de
proliferatie capaciteit van de lymfocyten. De veranderingen waren
zowel kwalitatief als kwantitatief vergelijkbaar met de veran
deringen die optraden onder invloed van intraveneuze toediening
van isoprenaline. Verder waren de effecten van tijdelijke aard.
120 Minuten na de toediening van insuline werden voor alle
parameters waarden genoteerd die niet significant afweken van
uitgangswaarden.
Terwijl dus acute stimulatie van het sympathische zenuwstelsel,
door exogene of endogene verhoging van de plasma catecholamine
spiegels, veranderingen teweeg brengt in de perifere lymfocyten
populatie, leek de endogene sympathische tonus hierop geen invloed
te hebben. Langdurige medicatie met de ß-blokkers bisoprolol,
Celiprolol en propranolol, had geen effect op de samenstelling
van de perifere lymfocyten populatie en ook niet op haar pro
liferatie capaciteit in vitro. Wel werd gevonden dat medicatie
met bisoprolol, Celiprolol en propranolol de door isoprenaline
gemedieerde veranderingen in de samenstelling van de perifere
lymfocyten populatie niet blokkeerde. Dit zou kunnen betekenen
dat dit fenomeen berust op een aspecifiek effect van isoprenaline.
Echter, het is ook mogelijk dat er een atypische ß-adrenoceptor
betrokken is bij de regulatie van de aantallen circulerende
lymfocyten.
Samenvattend kan worden gezegd dat in vitro studies aanwijzingen
leverden voor een immunosuppressieve functie van de ß2-adreno-
ceptor op lymfocyten. Echter, in vivo is de situatie veel com-
142
plexer. De endogene sympathische tonus lijkt van ondergeschikt
belang te zijn voor de immuun functie van lymfocyten, maar acute
stimulatie van het sympathische zenuwstelsel beïnvloedt wel
degelijk de perifere lymfocyten populatie. Zo verandert onder
invloed van exogene of endogene verhoging van de plasma cate
cholamine spiegels de samenstelling van de perifere lymfocyten
populatie, waarschijnlijk ten gevolge van een uitwisseling van
lymfocyten met secundaire lymfoide organen, en, wordt de pro
liferatie capaciteit van de circulerende lymfocyten onderdrukt.
Omdat de nieuwe lymfocyten een hogere ß2-adrenoceptor dichtheid
bezitten zouden ze gevoeliger kunnen zijn voor het immunosup-
pressieve effect van catecholamines. Dit kon echter niet worden
aangetoond.
Toekomstig onderzoek zal zich moeten richten op het mechanisme
en de fysiologische betekenis van de acute veranderingen in de
perifere lymfocyten populatie.
143
DANKSAGUNG
An dieser Stelle möchte Ich allen, die mir bei der Durchführung
dieser Doktorarbeit geholfen haben, recht herzlich danken:
Meinen Doktorvater Professor Dr. Otto-Erich Brodde für die sehr
interessante Aufgabenstellung sowie für die wohlwollende
Unterstützung während der Durchführung dieser Arbeit. Dr. Martin
Michel für zahlreiche wertvolle wissenschaftliche Diskussionen
zur Interpretation von Ergebnissen und bei Abfassung von
Manuskripten. Dr. Barbara Godau, Dr. Marek Wojcik und Frau Gisela
Pitzka für die Durchführung der invasiven Untersuchungen im
Kreislauflabor. Martina Michel für Bestimmungen von cAMP in
Lymphozyten sowie von a-Adrenozeptoren auf Thrombozyten und für
viele kleine Hilfestellungen bei Arbeiten, für die ich selbst
keine Zeit hatte.
Die Studenten der Medizin, Dieter Oefler und Gerd Krause, die im
Ramen ihrer Doktorarbeit viele Rezeptor-Bestimmungen mitdurch
geführt haben.
Matthias Krüger, für die Bestimmung von Plasma-Katecholamine
sowie die technische Überwachung aller Geräte, die ich bei dieser
Arbeit benutzt habe.
Dr. Jan Jacob Beckeringh ben ik erkentelijk voor zijn interesse,
suggesties en adviezen, en voor zijn steun bij het operationeel
maken van de "Pl-turnover" bepaling.
I thank Xiao Liang Wang and Nicky Deighton for the nice and
fruitful discussions concerning research and for "other inter
esting chats".
Christel Tietz und Hildegard Keller, die jederzeit bereit waren
sekretarielle Hilfestellung zu leisten.
Darüber hinaus möchte ich mich bei Jürgen Krusenberg, Hans-Jörg
Hundhausen, Mirjam Gnadt, Peter Klesse, Werner söhlmann, Matthias
Sundermann, Jörg Knapp und Sabine Jäger (Studenten der Medizin),
sowie bei Massud Khamssi, Petra Helming, Ellen Schäfer, Andrea
144
Broede, Susanne Bals, Angelika Tillmann und Ute Bolitz (Mitar
beiter der Abteilung) für die gute kollegiale Zusammenarbeit in
einer sehr angenehmen Atmosphäre bedanken.
Prof. Dr. H. Grosse-Wilde danke ich für sein Interesse an meiner
Arbeit und für seine Bereitschaft, mir Raum, Material und Personal
zur Isolierung von Lymphozyten Subpopulationen zur Verfügung zu
stellen. Frau Marion Happel möchte ich herzlich danken für ihre
Hilfe bei Isolierung und Bestimmung der Lymphozyten Subpopula
tionen, für die unzählige Blutentnahmen und für die Gast
freundschaft. Den Technischen Assistentinnen vom MLC Labor möchte
ich für die jederzeit sorgfältige Bearbeitung von angekündigten,
besonders aber von nicht angekündigten Blutproben danken.
145
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Metabolism 71: 187-192, 1990.
Graafsma, S.J., L.J.H. van Tits, P.H.G.M. Willems, M.P.C. Hectors,
J.F. Rodrigues de Miranda, J.J.H.H.M. de Pont and T. Thien.
ß2-adrenoceptor up-regulation in relation to cAMP production in
human lymphocytes after physical exercise. British Journal of
Clinical Pharmacology 30: 142S-144S, 1990.
Graafsma, S.J., M.P.C. Hectors, L.J.H. van Tits, J.F. Rodrigues
de Miranda and T. Thien. The relationship between adrenaline and
ß2-adrenoceptors on human lymphocytes. British Journal of Clinical
Pharmacology 30: 144S-147S, 199Q.
Van Tits, L.J.H., M.C. Michel, H.J. Motulsky, A.S. Maisel and
O.-E. Brodde. Cyclic AMP counteracts mitogen-induced inositol
phosphate generation and increases in intracellular Са++-соп-
centrations in human lymphocytes. Britisch Journal of Pharmacology
103: 1288-1294, 1991.
Wang, X.L., L.J.H. van Tits and N.M. Deighton. Agonist-specific
and non-specific in vitro desensitization of human lymphocyte
ß2-adrenoceptors. Asia Pacific Journal of Pharmacology 6: 89-97,
1991.
147
Graafsma, S.J., H. Wollersheim, H.T. Droste, M.A.G.J. ten Dam,
L.J.H. van Tits, J. Reyenga, J.F. Rodrigues de Miranda and Th.
Thien. Adrenoceptors on blood cells from patients with primary
Raynaud's phenomenon. Clinical Science 80: 325-331, 1991.
Brodde, O.-E., L.J.H. van Tits and M.C. Michel. Acute immuno
modulatory effects of ß-adrenoceptor agonists. In: "Adrenocep
tors: structure, mechanisms, function", Eds. E. Szabadi and CM.
Bradshaw, Birkhäuser Verlag, Basel, 237-244, 1991.
Van Tits, L.J.H. and S.J. Graafsma. Stress influences CD4+
lymphocyte counts. Immunology Letters 30: 141-142, 1991.
Maisei, A.S., D. Murray, S. Polizzi, H.J. Motulsky, O.-E. Brodde,
L.J.H. van Tits and M.C. Michel. Does verapamil act as an
immunomodulatory drug іл vivo? Immunopharmacology 22: 85-92,
1991.
Michel, M.C, L.J.H. van Tits, G. Trenn and O.-E. Brodde. Dis
sociation between phytohemagglutinin-stimulated generation of
inositol phosphates and Ca++ increases in human mononuclear
leukocytes. Biochemical Journal, submitted.
148
CURRICULUM VITAE
Berry van Tits werd geboren op 28 oktober 1961 te Boxmeer. In
1980 behaalde hij het VWO diploma aan het Elzendaalcollege te
Boxmeer. In datzelfde jaar begon hij met de studie Biologie aan
de Katholieke Universiteit van Nijmegen. In 1984 legde hij het
kandidaatsexamen af met hoofdvak Biologie en bijvak Geologie
(letter Big). Het doctoraalexamen, met Dierfysiologie (Prof.
Wendelaar Bonga) als hoofdrichting binnen het hoofdvak Biologie,
en met de bijvakken Ontwikkelingsbiologie der Dieren (Prof.
Denucé) en Gynaecologie en Verloskunde (Prof. Rolland, St.
Radboudziekenhuis), werd afgelegd in augustus 1987.
Van september 1987 tot december 1990 was hij werkzaam als
wetenschappelijk medewerker aan het "Institut für Nieren- und
Hochdruckkrankheiten" van het "Universitätsklinikum" te Essen.
Sinds juli 1991 is hij werkzaam aan de Vrije Universiteit van
Amsterdam bij de vakgroep Farmacochemie.
Hij is getrouwd met Elly Ermers. Zij hebben twee kinderen,
Elzemiek en Etienne.
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