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.

REFERENCES

1. Cryer P.E., 1980. Physiology and pathophysiology of the human

sympathoadrenal neuroendocrine system. N. Engl. J. Med. 303,

436-444.

2. Ahlquist R.P., 1948. A study of the adrenotropic receptors.

Am. J. Physiol. 153, 586-600.

3. Berthelsen S. and Pettinger W.A., 1977. A functional basis

for classification of oj-adrenergic receptors. Life Sci. 21,

595-606.

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

thomimetic amines. Nature 21A, 597-598.

6. Minneman K.P., 1988. ß-Adrenergic receptor subtypes. ISI Atl.

Sci. Pharmacol., 334-338.

7. Silverberg A.B., Shah S.D., Raymond M.W. and Cryer P.E.,

1978. Norepinephrine: hormone and neurotransmitter in man. Am.

J. Physiol. 234, E252-E256.

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) .

24. Burman K.D., Ferguson E.W., Djuh Y.-Y., Wartofsky L. and

Latham Κ., 1985. Beta receptors in peripheral mononuclear cells

increase acutely during exercise. Acta Endocrinol. 109, 563-568.

25. DeBlasiA., MaiselA.S., Feldman R.D., Ziegler M.G., Fratelli

9

M., DiLallo M., Smith D.A. , Lai C.-Y.C, Motulsky H. J. , 1986. 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. 63, 847-853.

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

lamines after mental arithmetic in normotensive volunteers. J.

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.

REFERENCES

1. Harden TK. Agonist-induced desensitization of the ß-adrenergic

receptor-linked adenylate cyclase. Pharmacological Reviews 1983;

35: 5-32.

2. Hertel С and Perkins JP. Receptor-specific mechanisms of

desensitization of ß-adrenergic receptor function. Molecular and

Cellular Endocrinology 1984; 37: 245-256.

3. Stiles GL, Carón MG and Lefkowitz RJ. ß-Adrenergic receptors:

biochemical mechanisms of physiological regulation. Physiological

Reviews 1984; 64: 661-743.

4. Lefkowitz RJ and Caron MG. Adrenergic receptors: molecular

mechanisms of clinically relevant regulation. Clinical Research

1985; 33: 395-406.

5. Lefkowitz RJ and Caron MG. Regulation of adrenergic receptor

function by phosphorylation. Journal of Molecular and Cellular

Cardiology 1986; 18: 885-895.

6. Sibley DR and Lefkowitz RJ. Biochemical mechanisms of

ß-adrenergic receptor regulation. ISI Atlas of Science Pharma­

cology 1988; 2: 66-70.

7. Kelleher DJ, Pessin JE, Ruoho AE and Johnson GL. Phorbol

ester induces desensitization of adenylate cyclase and phos­

phorylation of the ß-adrenergic receptor in turkey erythrocytes.

Proceedings of the National Academy of Sciences USA 1984; 81:

4316-4320.

8. Nambi P, Peters JR, Sibley DR and Lefkowitz RJ. Desensitization

of the turkey erythrocyte ß-adrenergic receptor in a cell free

system. Evidence that multiple protein kinases can phosphorylate

and desensitize the receptor. Journal of Biological Chemistry

1985; 260: 2165-2171.

9. Sibley DR, Nambi P, Peters JR and Lefkowitz RJ. Phorbol

diesters promote ß-adrenergic receptor phosphorylation and

26

adenylate cyclase desensitization in duck erythrocytes. Bio­

chemical and Biophysical Research Communications 1984; 121:

973-979.

10. Nishizuka Y. The role of protein kinase С in cell surface

signal transduction and tumour promotion. Nature 1984; 308:

693-698.

11. Brodde O-Ε, Beckeringh JJ and Michel MC. Human heart

ß-adrenoceptors: a fair comparison with lymphocyte ß-adreno-

ceptors? Trends in Pharmacological Sciences 1987; 8: 403-407.

12. Motulsky HJ and Insel PA. Adrenergic receptors in man. Direct

identification, physiologic regulation, and clinical alterations.

New England Journal of Medicine 1982; 307: 18-29.

13. Böyum A. Isolation of mononuclear cells and granulocytes from

human blood.

Scandinavian Journal of Clinical and Laboratory Investigations

1968; 21 (Suppl 97): 77-89.

14. Gilman AG. A protein binding assay for adenosine 3',5'-cyclic

monophosphate. Proceedings of the National Academy of Sciences

USA 1970; 67: 305-312.

15. Schwabe U and Ebert R. Different effects of lipolytic hormones

and phosphodiesterase inhibitors on cyclic 3',5'-AMP levels in

isolated fat cells. Naunyn-Schmiedeberg's Archives of Pharma­

cology 1982; 274: 287-298.

16. Brodde O-Ε, Brinkmann M, Schemuth R, O'Hara N and Daul A.

Terbutaline-induced desensitization of human lymphocyte

ß2-adrenoceptors. Accelerated restoration of ß-adrenoceptor

responsiveness by prednisone and ketotifen. Journal of Clinical

Investigation 1985; 76: 1096-1101.

17. Scatchard G. The attraction of proteins for small molecules

and ions. Annals of the New York Academy of Science 1949; 51:

660-672.

18. McPherson GA. Analysis of radioligand binding experiments:

a collection of computer programs for the IBM PC. Journal of

Pharmacological Methods 1985; 14: 213-228.

19. Lands AM, Arnold A, McAuliff JP, Luduena FP and Brown TG.

Differentiation of receptor systems activated by sympathomimetic

27

amines. Nature 1967; 214: 597-598.

20. Lands AM, Luduena FP and Buzzo HJ. Differentiation of receptors

responsive to isoproterenol. Life Science 1967; 6: 2241-2249.

21. Bilski AJ, Halliday SE, Fitzgerald JD and Wale JL. The

pharmacology of a ß2-selective adrenoceptor antagonist (ICI

118,551). Journal of Cardiovascular Pharmacology 1983 ; 5: 430-437.

22. Wang XL, Brinkmann M and Brodde 0-E. Selective labelling of

ßj^-adrenoceptors in rabbit lung membranes by (-) [3H]bisoprolol.

European Journal of Pharmacology 1985; 114: 157-165.

23. Brodde O-Ε, Daul A, Wellstein A, Palm D, Michel MC and

Beckeringh JJ. Differentiation of ß1- and ßj-adrenoceptor-mediated

effects in humans. American Journal of Physiology 1988; 254:

H199-H206.

24. Bourne HR and Melmon KL. Adenyl cyclase in human leukocytes:

Evidence for activation by separate ß-adrenergic and prostaglandin

receptors. Journal of Pharmacology and Experimental Therapeutics

1971; 178: 1-7.

25. Sandnes D, Gjerde I, Resnes M and Jacobsen S. Down-regulation

of surface beta-adrenoceptors on intact human mononuclear leu­

cocytes. Time-course and isoproterenol concentration dependence.

Biochemical Pharmacology 1987; 36: 1303-1311.

26. Bell JD, Buxton LO and Brunton LL. Enhancement of adenylate

cyclase activity in S49 lymphoma cells by phorbol esters. Journal

of Biological Chemistry 1985; 260: 2625-2628.

27. Bell JD and Brunton LL. Multiple effects of phorbol esters

on hormone-sensitive adenylate cyclase activity in S49 lymphoma

cells. American Journal of Physiology 1987; 252: E783-E789.

28. Sibley DR, Jeffs RA, Daniel K, Nambi Ρ and Lefkowitz RJ.

Phorbol diester treatment promotes enhanced adenylate cyclase

activity in frog erythrocytes. Archives of Biochemistry and

Biophysics 1986; 244: 373-381.

29. Yamashita A, Kurokawa T, Dan'ura T, Higashi К and Ishibashi

S. Induction of desensitization by phorbol ester to ß-adrenergic

agonist stimulation in adenylate cyclase system of rat reticu­

locytes. Biochemical and Biophysical Research Communications

1986; 138: 125-130.

28

30. Fishman PH, Sullivan M and Patel J. Down-regulation of protein

kinase С in rat glioma Сб cells: effects on the ß-adrenergic

receptor-coupled adenylate cyclase. Biochemical and Biophysical

Research Communications 1987; 144: 620-627.

31. Seamon К and Daly JW. Activation of adenylate cyclase by the

diterpene forskolin does not require the guanine nucleotide

regulatory protein. Journal of Biological Chemistry 1981; 256:

9799-9801.

32. Bell JD and Brunton LL. Enhancement of adenylate cyclase

activity in S49 lymphoma cells by phorbol esters. Journal of

Biological Chemistry 1986; 261: 12036-12041.

33. Johnson JA, Goka TJ and Clark RBJ. Phorbol ester-induced

augmentation and inhibition of epinephrine-stimulated adenylate

cyclase in S49 lymphoma cells. Journal of Cyclic Nucleotide and

Protein Phosphorylation Research 1986; 11: 199-215.

34. Langlois D, Saez J-M and Begeot M. The potentiating effects

of phorbol ester on ACTH-, cholera toxin-, and forskolin-induced

cAMP production by cultured bovine adrenal cells is not mediated

by the inactivation of α subunit of Gi protein. Biochemical and

Biophysical Research Communications 1987; 146: 517-523.

35. Jakobs KH, Bauer S and Watanabe Y. Modulation of adenylate

cyclase of human platelets by phorbol ester. European Journal of

Biochemistry 1985; 151: 425-430.

36. Katada T, Gilman AG, Watanabe Y, Bauer S and Jakobs КН.

Protein kinase С phosphorylates the inhibitory guanine

nucleotide-binding regulatory component and apparently suppresses

its function in hormonal inhibition of adenylate cyclase. European

Journal of Biochemistry 1985; 151: 431-437.

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.

REFERENCES

1. Aarons, R.D., A.S. Nies, J. Gal, L.R. Hegstrand, and P.B.

Molinoff. Elevation of ß-adrenergic receptor density in human

lymphocytes after propranolol administration. J. Clin. Invest.

65: 949-957, 1980.

2. Ahlborg, В., and G. Ahlborg. Exercise leukocytosis with and

55

without beta-adrenergic blockade. Acta. Med. Scand. 187: 241-246,

1987.

3. Anderson, J.L., J.F. Cardiquist, and E.H. Hammond. Deficient

natural killer cell activity in patients with idiopathic dilated

cardiomyopathy. Lancet 2: 1124-1127, 1982.

4. Beckeringh, J.J., O.-E. Brodde, A. Daul, M.C. Michel, and

X.L. Wang. Haemodynamic and lymphocyte ßj-adrenoceptor changes

during procaterol treatment in man (Abstract). Br. J. Pharmacol.

90, Supp2.:43P, 1987.

5. Bilski, A.J., S.E. Halliday, J.D. Fitzgerald, and J.L. Wale.

The pharmacology of a ß2-selective adrenoceptor antagonist (ICI

118,551). J. Cardiovasc. Pharmacol. 5: 430-437, 1983.

6. Bourne, H.R., L.M. Lichtenstein, K.L. Melmon, C.S. Henney,

Y. Weinstein, and G.M. Shearer. Modulation of inflammation and

immunity by cAMP: receptors for vasoactive hormones and mediators

of inflammation regulate many leucocyte functions. Science 184:

19-29, 1974.

7. Böyum, A. Isolation of mononuclear cells and granulocytes

from human blood. Scand. J. Clin. Lab. Invest. 21, Suppl. 97:

77-89, 1968.

8. Bradford, M.M. A rapid and sensitive method for the

quantitation of microgram quantities of protein utilizing the

principle of protein-dye binding. Anal. Biochem. 72: 248-254,

1976.

9. Brodde, O.-E. Bisoprolol (EMD 33512), a highly selective

ßj-adrenoceptor antagonist: in vitro and in vivo studies. J.

Cardiovasc. Pharmacol. 8, Suppl. 11: S29-S35, 1986.

10. Brodde, O.-E., J.J. Beckeringh, and M.C. Michel. Human heart

ß-adrenoceptors: a fair comparison with lymphocyte ß-adreno-

ceptors? Trends Pharmacol. Sci. 8: 403-407, 1987.

11. Brodde, O.-E., M. Brinkmann, R. Schemuth, N. O'Hara, and A.

Daul. Terbutaline-induced desensitization of human lymphocyte

ß2-adrenoceptors. Accelerated restoration of ß-adrenoceptor

responsiveness by prednisone and ketotifen. J. Clin. Invest. 76:

1096-1101, 1985.

12. Brodde, O.-E., A. Daul, and N. O'Hara. ß-Adrenoceptor changes

56

in human lymphocytes, induced by dynamic exercise. Wau-

nyn-Schmiedeberg's Arch. Pharmacol. 325: 190-192, 1984. (Erratum:

Waunyn-Schmiedeberg's Arch. Pharmacol. 328: 362, 1985).

13. Brodde, O.-E., A. Daul, N. Stuka, N. O'Hara, and U. Borchard.

Effects of ß-adrenoceptor antagonist administration on

ß2-adrenoceptor density in human lymphocytes. The role of "the

intrinsic sympathomimetic activity". Naunyn Schmiedeberg's Arch.

Pharmacol. 328: 417-422, 1985.

14. Brodde, O.-E., A.E. Daul, X. L. Wang, M.C. Michel, and 0.

Galal. Dynamic exercise-induced increase in lymphocyte beta-2-

adrenoceptors: abnormality in essential hypertension and its

correction by antihypertensives. Clin. Pharmacol. Ther. 41:

371-379, 1987.

15. Brodde, O.-E., A. Daul, A. Wellstein, D. Palm, M.C. Michel,

and J.J. Beckeringh. Differentiation of ß ^ and

ß2-adrenoceptor-mediated effects in humans. Алі. J. Physiol. 254:

H199-H206, 1988.

16. Burman K.D., E.W. Ferguson, Y.-Y. Djuh, L. Wartofsky, and K.

Latham. Beta receptors in peripheral mononuclear cells increase

acutely during exercise. Acta. Endocrinol. 109: 563-568, 1985.

17. Butler, J., J.G. Kelly, K. O'Malley, and F. Pidgeon.

ß-Adrenoceptor adaptation to acute exercise. J. Physiol. Lond.

344: 113-118, 1983.

18. Crary, В., M. Borysenko, D.C. Sutherland, I. Kutz, J.Z.

Borysenko, and H. Benson. Decrease in mitogen responsiveness of

mononuclear cells from peripheral blood after epinephrine

administration in humans. J. Immunol. 130: 694-697, 1983.

19. Crary, В., S.L. Hauser, M. Borysenko, I. Kutz, C. Hoban, K.A.

Ault, H.L. Weiner, and H. Benson. Epinephrine-induced changes in

the distribution of lymphocyte subsets in peripheral blood of

humans. J. Immunol. 131: 1178-1181, 1983.

20. Dailey, M.O., J. Schreurs, and H. Schulman. Hormone receptors

on cloned Τ lymphocytes. Increased responsiveness to histamine,

prostaglandins, and ß-adrenergic agents as a late stage event in

Τ cell activation. J. Immunol. 140: 2931-2936, 1988.

21. DeBlasi, Α., M. Lipartiti, H.J. Motulsky, P.A. Insel, and M.

57

Fratelli. Agonist-induced redistribution of ß-adrenergic

receptors on intact human mononuclear leukocytes. Redistributed

receptors are nonfunctional. J. Сііл. Endocrinol. Metab. 63:

847-853, 1986.

22. DeBlasi, Α., A.S. Maisel, R.D. Feldman, M.G. Ziegler, M.

Fratelli, M. DiLallo, D.A. Smith, C.-Y.C. Lai, and H.J. Motulsky.

In vivo regulation of ß-adrenergic receptors on human mononuclear

leucocytes: assessraent of receptor number, location, and function

after posture change, exercise, and isoproterenol infusion. J.

Clin. Endocrinol. Metab. 63: 847-53, 1986.

23. Deighton, N.M., L.J.H. van Tits, X.-L. Wang, andO.-E. Brodde.

In vitro studies on homologous and heterologous desensitization

of human lymphocyte ß2-adrenoceptors (Abstract) . Br. J. Pharmacol.

96: 126P, 1989.

24. Durant, S. In vivo effects of catecholamines and gluco­

corticoids on mouse thymic cAMP content and thymolysis. Cell.

Immunol. 102: 136-143, 1986.

25. Eckstein, R., W. Mempel, and H.D. Bolte. Reduced suppressor

cell activity in congestive cardiomyopathy and in myocarditis.

Circulation 65: 1224-1229, 1982.

26. Feldman, R.D., G.W. Hunninghake, and W.L. McArdle.

ß-Adrenergic receptor-mediated suppression of interleukin 2

receptors inhuman lymphocytes. J. Immunol. 139: 3355-3359, 1987.

27. Feiten, D.L., S.Y. Feiten, D.L. Bellinger, S.L. Carlson, K.D.

Ackerman, K.S. Madden, J.A. Olschowki, and S. Livnat. Norad­

renergic sympathetic neural interactions with the immune system:

structure and function. Immunol. Rev. 100: 225-260, 1987.

28. Foster, N.K., J.B. Martyn, R.E. Rangno, J.C. Hogg, and R.L.

Pardy. Leukocytosis of exercise: role of cardiac output and

catecholamines. J. Appi. Physiol. 61: 2218-2223, 1986.

29. Graafsma S.J., B. van Tits, B. Westerhof, R. van Valderen,

J.W.M. Lenders, J.F. Rodrigues de Miranda, and Th. Thien.

Adrenoceptor density on human blood cells and plasma catechol­

amines after mental arithmetic in normotensive volunteers. J.

Cardiovasc. Pharmacol. 10, Suppl 4: S107-S109, 1987.

30. Harden, Т.К. Agonist-induced desensitization of the

58

ß-adrenergic receptor-linked adenylate cyclase. Pharmacol. Rev.

35: 5-32, 1983.

31. Keller, S.E., J.M. Weiss, S.J. Schleiffer, N.E. Miller, and

M. Stein. Suppression of immunity by stress: effect of a graded

series of stressors on lymphocyte stimulation in the rat. Science

Wash. DC 213: 1397-1400, 1981.

32. Keller, S.E., J.M. Weiss, S.J. Schleiffer, N.E. Miller, and

M. Stein. Stress-induced suppression of immunity in adrenalec-

tomized rats. Science Wash. DC 221: 1301-1304, 1983.

33. Khan, M.M., K.L. Melmon, CG. Fathman, B. Hertel-Wulff, and

S. Strober. The effects of autacoids on cloned murine lymphoid

cells: modulation of II 2 secretion and the activity of natural

suppressor cells. J. Immunol. 134: 4100-4106, 1985.

34. Khan, M.M., P. Sansoni, E.D. Silverman, E.G. Engleman, and

K.L. Melmon. ß-Adrenergic receptors on human suppressor, helper,

and cytolytic lymphocytes. Biochem. Pharmacol. 35: 1137-1143,

1986.

35. Knowlton, K.U., A. Rearden, M.C. Michel, P.A. Insel, and A.S.

Maisel. Lymphocyte subsets in heart failure following acute

sympathetic stimulation (Abstract). Circulation 78, Suppl 2:

11-165, 1988.

36. Landmann, R.M.Α., E. Bürgisser, M. Wesp, and F.R. Bühler.

Beta-adrenergic receptors are different in subpopulations of

human circulating lymphocytes. J. Recept. Res. 4: 37-50, 1984.

37. Landmann, R.M.Α., F.B. Müller, С. Perini, M. Wesp, P. Erne,

and F.R. Bühler. Changes of immunoregulatory cells induced by

psychological and physical stress: relationship to plasma

catecholamines. Clin. Exp. Immunol. 58: 127-35, 1984.

38. Lands, A.M., A. Arnold, J.P. McAuliff, F.P. Luduena, andT.G.

Brown. Differentiation of receptor systems activated by sympa­

thomimetic amines. Nature Lond. 214: 597-598, 1967.

39. Laudenslager, M.L., S.M. Ryan, R.C. Drugan, R.L. Hyson, and

S.F. Maier. Coping and immunosuppression: inescapable but not

escapable shock suppresses lymphocyte proliferation. Science

Wash. DC 221: 568-570, 1983.

40. Maisel, A.S., and H.J. Motulsky. Receptor distribution does

59

not accompany terbutaline-induced down regulation of

beta-adrenergic receptors on human mononuclear leukocytes. Clin.

Pharm. Ther. 42: 100-106, 1987.

41. Michel, M.C., A. Pingsmann, J.J. Beckeringh, H.-R. Zerkowski,

N. Doetsch, and O.-E. Brodde. Selective regulation of R^ and

ß2-adrenoceptors in the human heart by chronic ß-adrenoceptor

antagonist treatment. Br. J. Pharmacol. 94: 685-692, 1988.

42. Michel, M.C., A. Pingsmann, M. Nohlen, U. Siekmann, and O.-E.

Brodde. Decreased myometrial ß-adrenoceptors in women receiving

ß2-adrenergic tocolytic therapy: correlation with lymphocyte

ß-adrenoceptors. Clin. Pharmacol. Ther. 45: 1-8, 1989.

43. Monjan, A.A., and M.I. Collector. Stress-induced modulation

of the immune response. Science Wash. DC 196: 307-308, 1977.

44. Motulsky, H.J., E.M.S. Cunningham, A. DeBlasi, and P.A. Insel.

Desensitization and redistribution of ß-adrenergic receptors on

human mononuclear leukocytes. Am. J. Physiol. 250: (.Endocrinol.

Metah. 13): E583-E590, 1986.

45. Motulsky, H.J., and Insel, P.A. Adrenergic receptors in man.

Direct identification, physiologic regulation, and clinical

alterations. N. Engl. J. Med. 307: 18-29, 1982.

46. Scatchard, G. The attraction of proteins for small molecules

and ions. Алл. NY Acad. Sci. 51: 660-672, 1949.

47. Strittmatter, W.J., F. Hirata, and J. Axelrod. Regulation of

the ß-adrenergic receptor by methylation of membrane phos­

pholipids. In: Advances іл Cyclic Nucleotide and Protein Phos­

phorylation Research, edited by J.E. Dumant, P. Greengard, and

G.A. Robison. New York: Raven, 1981, vol. 14, p. 83-91.

48. Tohmeh, J.F., and P.E. Cryer. Biphasic adrenergic modulation

of ß-adrenergic receptors in man. Agonist-induced early increment

and late decrement in ß-adrenergic receptor number. J. Clin.

Invest. 65: 836-840, 1980.

49. Wang, X.L., and O.-E. Brodde. Identification of a homogeneous

class of ß2-adrenoceptors in human platelets by (-)-125I-iodo-

pindolol binding. J. Cyclic. Nucleotide Protein Phosphorylation

Res. 10: 439-450, 1985.

50. Wellstein, Α., G.G. Beiz, and D. Palm. Beta adrenoceptor

60

subtype binding activity in plasma and beta blockade by propranolol

and beta-1 selective bisoprolol in humans. Evaluation with

Schild-plots. J. Pharmacol.Exp. Ther. 246: 328-337, 1988.

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?

75

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.

1980;65:836-40.

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.

9. Brodde O-Ε, Daul A, Wellstein A, Palm D, Michel MC, Beckeringh

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

76

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.

17. Khan MM, Melmon KL, Fathman CG, Hertel-Wulff B, Strober S.

The effects of autacoids on cloned murine lymphoid cells:

modulation of II 2 secretion and the activity of natural suppressor

cells. J Immunol. 1985;134:4100-6.

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

ß2-adrenoceptors. Accelerated restoration of ß-adrenoceptor

responsiveness by prednisone and ketotifen. J Clin Invest.

1985;76:1096-1101.

21. Scatchard G. The attraction of proteins for small molecules

and ions. Ann NY Acad Sci. 1949;51:660-672.

22. Middeke M, Lohnmöller G, Remien J, Kirzinger S, Holzgreve H.

77

Acute regulation of lymphocyte ß-adrenoceptor activity in

pheochromocytoma. Klin Wochenschr. 1988;66:187-9.

23. Strasser RH, Stiles GL, Lefkowitz RJ. Translocation and

uncoupling of the ß-adrenergic receptor in rat lung after

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

lymphocytes. Biochem Pharmacol. 1986;35:1137-1143.

28. Maisel AS, Fowler P, Rearden A, Motulsky HJ, Michel MC. A

new method for isolation of human lymphocyte subsets reveals

differential regulation of ß-adrenergic receptors by terbutaline

treatment. Clin Pharmacol Ther. 1989;46:429-39.

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.

References

Alcover, Α., Ramarli, D., Richardson, N.E., Chang, H.-C. and

Reinherz, E.L. (1987). Functional and molecular aspects of human

Τ lymphocyte activation via ТЗ-Ti and Til pathways. Immunol. Rev.

95: 5-36.

Aussei, С , Mary, D. , Peyron, J.-F., Pelassy, С , Ferrua, В.

and Fehlman, M. (1988). Inhibition and activation of interleukin

2 synthesis by direct modification of guanosine triphospha-

te-binding proteins. J_¡_ Immunol. 140: 215-220.

Berridge, M.J. and Irvine, R.F. (1989). Inositol phosphates

and cell signalling. Nature 341: 197-205.

Bilski, A.J., Halliday, S.E., Fitzgerald, J.D. and Wale, J.L.

(1983) . The pharmacology of a ßj-selective adrenoceptor antagonist

(ICI 118,551). J. Cardiovasc. Pharmacol. 5: 430-437.

Böyum, A. (1968). Isolation of mononuclear cells and

granulocytes from human blood. Scand. J. Clin. Lab. Invest. 21:

77-89.

Bourne, H.R., Lichtenstein, L.M., Melmon, K.L., Henney, C.S.,

Weinstein, Y. and Shearer, G.M. (1974). Modulation of inflammation

and immunity by cAMP. Science 184:19-29.

Brodde, O.-E., Beckeringh, J.J. and Michel, M.C. (1987).

Human heart ß-adrenoceptors: a fair comparison with lymphocyte

ß-adrenoceptors? Trends Pharmacol. Sci. 8: 403-407.

Brodde, O.-E., Brinkmann, M., Schemuth, R., O'Hara, N. and

Daul, A. (1985). Terbutaline-induced desensitization of human

lymphocyte ß2-adrenoceptors. Accelerated restoration of

ß-adrenoceptor responsiveness by prednosine and ketotifen. J.

Clin. Invest. 26: 1096-1101.

Gambier, J.C., Justement, L.B., Newell, M.K., Chen, Z.Z.,

99

Harris, L.K., Sandoval, V.M., Klemsz, M.J. and Ransom, J.T.

(1987) . Transmembrane signals and intracellular "second mes­

sengers" in the regulation of quiescent B-lymphocyte activation.

Immunol. Rev. 95; 37-57.

Chen, Z.Z., Coggeshall, K.M. and Gambier, J.C. (1986).

Translocation of protein kinase С during membrane immunoglobu-

lin-mediated transmembrane signalling in В lymphocytes. J.

Immunol. 136: 2300-2304.

Dailey, M.O., Schreurs, J. and Schulman, H. (1988). Hormone

receptors on cloned Τ lymphocytes. Increased responsiveness to

histamine, prostaglandins, and ß-adrenergic agents as a late

stage event in Τ cell activation. ¡L. Immunol. 140: 2931-2936.

Dooley, D.J., Bittiger, Η. and Reymann, N.C. (1986). CGP

20712A: an useful tool for quantitating ßj- and ß2-adrenoceptors.

Eur. J. Pharmacol. 130: 137-139.

Durant, S. (1986). In vivo effects of catecholamines and

glucocorticoids on mouse thymic cAMP content and thymolysis.

Cell. Immunol. 102: 136-143.

Feldman, R.D., Hunninghake, G.W. and McArdle, W.L. (1987).

ß-Adrenergic receptor-mediated suppression of interleukin 2

receptors in human lymphocytes. ¡L. Immunol. 139: 3355-3359.

Feiten, D.L., Feiten, S.Y., Bellinger, D.L., Carlson, S.L.,

Ackerman, K.D., Madden, K.S., Olschowki, J.A. and Livnat, S.

(1987). Noradrenergic sympathetic neural interactions with the

immune system: structure and function. Immunol. Rev. 100: 225-260.

Fredholm, B.B., Jondal, M. and Nordstedt, C. (1987). The

adenosine receptor mediated accumulation of cyclic AMP in Jurkat

cells is enhanced by a lectin and by phorbol esters. Biochem.

Biophys. Res. Commun. 145: 344-349.

Gelfand, E.W., Mills, G.B., Cheung, R.K., Lee, J.W.W, and

Grinstein, S. (1987). Transmembrane ion fluxes during activation

of human Τ lymphocytes: role of Ca2+, Na

+/H

+ exchange and phos­

pholipid turnover. Immunol. Rev. 95: 59-87.

Gray, L.S., Huber, K.S., Gray, M.C., Hewlett, E.L. and

Engelhard, V.H. (1989). Pertussis toxin effects on Τ lymphocytes

are mediated through CD3 and not by pertussis toxin catalyzed

100

modification of a G protein. ¡L. Immunol. 142; 1631-1638.

Gupta, S. (1989). Mechanisms of transmembrane signalling in

human Τ cell activation. Mol. Cell. Biochem. 91; 45-50.

Hadden, J.W. (1988). Transmembrane signals in the activation

of Τ lymphocytes by mitogenic antigens. Immunol. Today 9: 235-239.

Imboden, J.В., Shoback, D.M., Pattison, G. and Stobo, J.D.

(1986). Cholera toxin inhibits the T-cell antigen receptor-me­

diated increases in inositol triphosphate and cytoplasmic free

calcium. Proc. Natl. Acad. Sci. USA 83: 5673-5677.

Johnson, K.W., Davis, B.H. and Smith, K.A. (1988). cAMP

antagonizes interleukin 2-promoted T-cell cycle progression at

a discrete point in early G1. Proc. Natl. Acad. Sci. USA 85:

6072-6076.

Kammer, G.M. (1988) . The adenylate cyclase cAMP-protein kinase

A pathway and regulation of immune response. Immunol. Today 9:

222-228.

King, S.L. (1988). An assessment of phosphoinositide

hydrolysis in antigenic signal transduction in lymphocytes.

Immunol. 65: 1-7.

Lands, A.M., Arnold, Α., McAuliff, J.P., Luduena, F.P. and

Brown, T.G. (1967). Differentiation of receptor systems activated

by sympathomimetic amines. Nature (London) 214: 597-598.

Lerner, Α., Jacobson, В. and Miller, R.A. (1988). Cyclic AMP

concentrations modulate both calcium influx and hydrolysis of

phosphatidylinositol phosphates in mouse Τ lymphocytes. J.

Immunol. 140: 936-940.

Linch, D.C., Wallace, D.L. and O'Flynn, K. (1987). Signal

transduction in human Τ lymphocytes. Immunol. Rev. 95: 137-159.

Macintyre, E.A., Tatham, P.E.R., Abdul-Gaffar, R. and Linch,

D.C. (1988) . The effects of pertussis toxin on human Τ lymphocytes.

Immunol. 64: 427-432.

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.

J. Immunol. 139: 1979-1984.

Minneman, K.P. and Johnson, R.D. (1984). Characterization of

alpha-1 adrenergic receptors linked to [3H]-inositol metabolism

in rat cerebral cortex. J . Pharmacol. Exp. Ther. 230: 317-323.

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.

Sommermeyer, H. and Resch, K. (1990). Pertussis toxin

B-subunit-induced Ca++-fluxes in Jurkat human lymphoma cells: the

action of long-term pre-treatment with cholera and pertussis

holotoxins. Cell. Signalling 2: 115-128.

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.

Sugiura, T. and Waku, K. (1984). Enhanced turnover of ara-

chidonic acid-containing species of phosphatidylinositol and

phosphatidic acid of concanavalin Α-stimulated lymphocytes.

Biochim. Biophys. Acta 796: 190-198.

Taylor, M.V., Metcalfe, J.C., Hesketh, T.R., Smith, G.A. and

Moore, J.P. (1984) . Mitogens increase phosphorylation of phos-

phoinositides in thymocytes. Nature (London) 312: 462-465.

Tsien, R.Y., Pozzan, T. and Rink, T.J. (1982). T-cell mitogens

cause early changes in cytoplasmic free Ca2 + and membrane potential

in lymphocytes. Nature (London) 295: 68-71.

Windebank, K.P., Abraham, R.T., Powis, G. , Olsen, R.A., Barna,

T.J. and Leibson, P.J. (1988). Signal transduction during human

natural killer cell activation: inositol phosphate generation

and regulation by cyclic AMP. JN Immunol. 141: 3951-3957.

Wolff, C.H.J, and Akerman, K.E.O. (1982). Concanavalin A

binding and Ca2 + fluxes in rat spleen cells. Biochim. Biophys.

Acta. 693: 315-319.

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.

REFERENCES

1. Feiten DL, Feiten SY, Bellinger DL, Carlson SL, Ackerman KD,

Madden KS, Olschowki JA and Livnat S. Noradrenergic sympathetic

neural interactions with the immune system: structure and

function. Immunol Rev 1987;100:225-60.

2. Brodde O-Ε, Beckeringh JJ and Michel MC. Human heart

ß-adrenoceptors: a fair comparison with lymphocyte ß-adreno-

ceptors? Trends Pharmacol Sci 1987;8:403-7.

3. Kammer GM. The adenylate cyclase cAMP-protein kinase A pathway

and regulation of immune response. Immunol Today 1988;9:222-8.

4. Van Tits LJH, Michel MC, Motulsky HJ, Maisel AS and Brodde

O-Ε. Cyclic AMP counteracts mitogen-induced inositol phosphate

generation and increases in intracellular Ca++-concentrations in

human lymphocytes. Br J Pharmacol 1990;103:1288-1294.

5. Landmann RMA, Müller FB, Perini С, Wesp M, Erne Ρ and Bühler

FR. Changes of immunoregulatory cells induced by psychological

and physical stress: relationship to plasma catecholamines. Clin

Exp Immunol 1984;58:127-35.

6. Foster NK, Martyn JB, Rangno RE, Hogg JC and Pardy RL.

Leukocytosis of exercise: role of cardiac output and catechol­

amines. J Appi Physiol 1986;61:2218-2223.

7. Ahlborg В and Ahlborg G. Exercise leukocytosis with and without

beta-adrenergic blockade. Acta Med Scand 1970;187:241-246.

8. Van Tits LJH, Daul A, Bauch HJ, Grosse-Wilde H, Happel M,

Michel MC and Brodde O-Ε. Effects of insulin-induced hypoglycemia

on ß2-adrenoceptor density and proliferative responses of human

lymphocytes. J Clin Endocrinol Metab 1990;71:187-92.

115

9. 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 (Endocrinol Metab) 1990;E191-E202.

10. Crary B, Hauser SL, Borysenko M, Kutz I, Hoban C, Ault KA,

Weiner HL and Benson H. Epinephrine-induced changes in the

distribution of lymphocyte subsets in peripheral blood of humans.

J Immunol 1983;131:1178-1181.

11. Brodde 0-E, Schemuth R, Brinkmann M, Wang XL, Daul A and

Borchard U. ß-Adrenoceptor antagonists (non-selective as well as

ß^selective) with partial agonistic activity decrease ß2-adre-

noceptor density in human lymphocytes. Evidence for a ß2-agonistic

component of the partial agonistic activity.

Naunyn-Schmiedeberg's Arch Pharmacol 1986;333:130-138.

12. Brodde O-Ε, Daul A, Wellstein A, Palm D, Michel MC and

Beckeringh JJ. Differentiation of ßj - and ß2-adrenoceptor-mediated

effects in humans. Am J Physiol 254 (Heart Circ Physiol 23)

1988:H199-H206.

13. Böyum A. Isolation of mononuclear cells and granulocytes from

human blood. Scand J Clin Lab Invest 1968;21 [Suppl 97]:77-89.

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.

References

1 Besedovsky H and Sorkin E. Network of immune-neuroendocrine

interactions. Clin Exp Immunol 1977;27:1-12.

2 Dunn AJ. Nervous system-immune system interactions: an

overview. J Receptor Res 1988;8:589-607.

3 Shanahan F and Anton P. Neuroendocrine modulation of the

immune system. Digest Dis Sci 1988;33:41S-49S.

4 O'Dorisio MS and Panerai A (eds). Neuropeptides and immu-

nopeptides: messengers in a neuroimmune axis. Ann Ν Y Acad Sci

1990;594:1-499.

5 Bourne HR, Lichtenstein LM, Melmon KL, Henney CS, Weinstein

Y and Shearer GM. Modulation of inflammation and immunity by

cAMP: receptors for vasoactive hormones and mediators of

128

inflammation regulate many leucocyte functions. Science

1974;184:19-29.

6 Feiten DL, Feiten SY, Bellinger DL, Carlson SL, Ackerman KD,

Madden KS, Olschowki JA and Livnat S. Noradrenergic sympathetic

neural interactions with the immune system: structure and

function. Immunol Rev 1987;100:225-260.

7 Kammer GM. The adenylate cyclase cAMP-protein kinase A pathway

and regulation of immune response. Immunol Today 1988;9:222-228.

8 Feldman RD, Hunninghake GW and McArdle WL. ß-Adrenergic

receptor-mediated suppression of interleukin 2 receptors in human

lymphocytes. J Immunol 1987;139:3355-3359.

9 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.

10 Johnson KW, Davis BH and Smith KA. CAMP antagonizes interleukin

2-promoted T-cell cycle progression at a discrete point in early

G1. Proc Natl Acad Sci USA 1988;85:6072-6076.

11 Monjan AA and Collector MI. Stress-induced modulation of the

immune response. Science 1977;196:307-308.

12 Laudenslager ML, Ryan SM, Drugan RC, Hyson RL and Maier SF.

Coping and immunosuppression: inescapable but not escapable shock

suppresses lymphocyte proliferation. Science 1983;221:568-570.

13 Keller SE, Weiss JM, Schleifer SJ, Miller NE and Stein M.

Stress-induced suppression of immunity in adrenalectomized rats.

Science 1983;221:1301-1304.

14 Landmann RMA, Müller FB, Perini С, Wesp M, Erne Ρ and Bühler

FR. Changes of immunoregulatory cells induced by psychological

and physical stress: relationship to plasma catecholamines. Clin

Exp Immunol 1984;58:127-135.

15 Khansari DN, Murgo AJ and Faith RE. Effects of stress on the

immune system. Immunol Today 1990;11:170-175.

16 Ferry A, Picard F, Duvallet A, Weill В and Rieu M. Changes

in blood leucocyte populations induced by acute maximal and

chronic submaximal exercise. Eur Appi Physiol 1990;59:435-442.

17 Van Tits LJH, Daul A, Bauch HJ, Grosse-Wilde H, Happel M,

Michel MC and Brodde O-Ε. Effects of insulin-induced hypoglycemia

129

on ß2-adrenoceptor density and proliferative responses of human

lymphocytes. J Clin Endocrinol Metab 1990;71:187-192.

18 Crary B, Borysenko M, Sutherland DC, Kutz I, Borysenko JZ

and Benson H. Decrease in mitogen responsiveness of mononuclear

cells from peripheral blood after epinephrine administration in

humans. J Immunol 1983;130:694-697.

19 Nordin AA and Proust JJ. Signal transduction mechanisms in

the immune system: potential implication in immunosenescence.

Endocrinol Metab Clin 1987;16:919-945.

20 Zanders ED. Lymphocyte receptors and transmembrane signalling

Тл Cohen and Houslay (eds): Molecular mechanisms of transmembrane

signalling. Elsevier Science Publishers BV, 1986:411-427.

21 Hadden JW. Transmembrane signals in the activation of Τ

lymphocytes by mitogenic antigens. Immunol Today 1988;9:235-239.

22 Crary B, Hauser SL, Borysenko M, Kutz I, Hoban C, Ault KA,

Weiner HL and Benson H. Epinephrine-induced changes in the

distribution of lymphocyte subsets in peripheral blood of humans.

J Immunol 1983;131:1178-1181.

23 Van Tits LJH, Michel MC, Grosse-Wilde Η, Happel M, Eigler

F-W, Soliman A and Brodde 0-E. Catecholamines increase lymphocyte

ß2-adrenergic receptors via a ß2-adrenergic, spleen-dependent

process. Am J Physiol (Endocrinol Metab) 1990;E191-E202.

24 Rabinovitch PS, June CH, Grossmann A and Ledbetter JA.

Heterogeneity among Τ cells in intracellular free calcium

responses after mitogen stimulation with PHA or anti-CD3. Sim­

ultaneous use of indo-1 and immunofluorescence with flow cyto­

metry. J Immunol 1986;137:952-961.

25 Adrenergic involvement in control of lymphocyte immune

function (Chapter 6, this thesis).

26 Böyum A. Isolation of mononuclear cells and granulocytes from

human blood. Scand J Clin Lab Invest 1968;21 [Suppl 97]:77-89.

27 Van Tits LJH, Michel MC, Motulsky HJ, Maisel AS and Brodde

0-E. Cyclic AMP counteracts mitogen-induced inositol phosphate

generation and increases in intracellular Ca++-concentrations in

human lymphocytes. Br J Pharmacol 1990;103:1288-1294.

28 Reinherz EL and Schlossman SF. Regulation of the immune

130

response: inducer and suppressor T-lymphocyte subsets in human

beings. N Engl J Med 1980;303:370-373.

29 Pabst R and Binns RM. Heterogeneity of lymphocyte homing

physiology: several mechanisms operate in the control of migration

to lymphoid and non-lymphoid organs in vivo. Immunol Rev

1989;108:83-109.

30 Pabst R. The spleen in lymphocyte migration. Immunol Today

1988;9:43-45.

31 Yednock TA and Rosen SD. Lymphocyte homing. Adv Immunol

1989;44:313-378.

32 Butcher EC. Cellular and molecular mechanisms that direct

leukocyte traffic. Am J Pathol 1990;136:3-11.

33 Albeda SM and Buck CA. Integrine and other cell adhesion

molecules. FASEB J 1990;4:2868-2880.

34 Khan MM, Sansoni Ρ, Silverman ED, Engleman EG and Melmon KL.

Beta-adrenergic receptors on human suppressor, helper, and

cytolytic lymphocytes. Biochem Pharmacol 1986;35:1137-1142.

35 Nishizuka Y. The role of protein kinase С in cell surface

signal transduction and tumour promotion. Nature 1984; 308:

693-698.

36 Kikkawa U and Nishizuka Y. The role of protein kinase С in

transmembrane signalling. Annu Rev Cell Biol 1986;2:149-178.

37 Blalock JE, Harbour-McMenamin D and Smith EM. Peptide hormones

shared by the neuroendocrine and immunologic systems. J Immunol.

1985;135:858s-861s.

38 Paul WE. Pleiotropy and redundancy: Τ cell-derived lymphokines

in the immune response. Cell 1989;57:521-524.

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

LIST OP PUBLICATIONS

Levels, P.J., L.J.H. van Tits and J.M. Denucé. The effect of the

presence of adult fishes, gonad homogenates, and embryo

homogenates on the dispersion-reaggregation phase during early

embryonic development of the annual fish Nothobranchius kort-

hausae. Journal of Experimental Zoology 240: 259-264, 1986.

Graafsma, S.J., L.J.H. van Tits, B. Westerhof, R. van Valderen,

J.W.M. Lenders, J.F. Rodrigues De Miranda and Th. Thien. Adre­

noceptor density on human blood cells and plasma catecholamines

after mental arithmetic in normotensive volunteers. Journal of

Cardiovascular Pharmacology 10 (suppl. 4): S107-S109, 1987.

Graafsma, S.J., L.J.H. van Tits, J.F. Rodrigues De Miranda and

Th. Thien. Kinetics of (-)125 iodocyanopindolol binding to intact

human mononuclear cells. Journal of Receptor Research 8: 773-785,

1988.

Graafsma, S.J., J.W.M. Lenders, J.H. Peters, L.J. van Tits, G.F.

Pieters, J.F. Rodrigues De Miranda and Th. Thien. Role of

adreneline in the short-term upregulation of beta-adrenoceptors

in essential hypertensive and adrenalectomized females. Journal

of Hypertension 6 (suppl. 4): S578-S580, 1988.

Van Tits, L.J.H., H. Grosse-Wilde, M. Happel, A.M. Khalifa and

O.-E. Brodde. ß-Adrenoceptor changes in leucocyte subpopulations

after isoprenaline-infusion in healthy volunteers. British

Journal of Clinical Pharmacology 27 (S): 660P-661P, 1989.

Graafsma, S.J., L.J. van Tits, P. van Heijst, J. Reyenga, J.W.M.

Lenders, J.F. Rodrigues De Miranda and TH. Thien. Adrenoceptors

on blood cells in patients with essential hypertension before

and after mental stress. Journal of Hypertension 7: 519-524,

1989.

Graafsma, S.J., L.J. van Tits, P. van Heijst, J. Reyenga, J.F.

Rodrigues De Miranda and Th. Thien. Effects of isometric exercise

on blood cell adrenoceptors in essential hypertension. Journal

of Cardiovascular Pharmacology 14: 598-602, 1989.

146

Van Tits, L.J.H., A. Daul, Η. Grosse-Wilde and O.-E. Brodde.

Cyclic AMP antagonizes mitogen-induced accumulation of inositol

phosphates in human peripheral mononuclear leucocytes in vitro.

British Journal of Clinical Pharmacology 30: 148S-149S, 1990.

Van Tits, L.J.H., M.C. Michel, H. Grosse-Wilde, M. Happel, F.-W.

Eigler, A. Soleman and O.-E. Brodde. Catecholamines increase

lymphocyte ß2-adrenergic receptors via a ß2-adrenergic,

spleen-dependent process. American Journal of Physiology 258:

E191-E202, 1990.

Van Tits, L.J.H., A. Daul, H.J. Bauch, H. Grosse-Wilde, M. Happel,

M.C. Michel and O.-E. Brodde. Effects of insulin-induced hypo­

glycemia on ß2-adrenoceptor density and proliferative responses

of human lymphocytes. Journal of Clinical Endocrinology and

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.

149