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The Journal of Clinical Endocrinology & MetabolismCopyright © 2000 by The Endocrine Society

Disturbed Neuroendocrine-Immune Interactions inChronic Fatigue Syndrome*

ANNEMIEKE KAVELAARS, WIETSE KUIS, LIDEWIJ KNOOK, GERBEN SINNEMA,AND COBI J. HEIJNEN

Departments of Pediatric Immunology (A.K., W.K., L.K., C.J.H.) and Psychology (G.S.),Wilhelmina Children’s Hospital of the University Medical Center Utrecht, 3584 EA Utrecht,The Netherlands

Vol. 85, No. 2

Printed in U.S.A.

ABSTRACT

The present study was designed to investigate the interactionbetween neuroendocrine mediators and the immune system inchronic fatigue syndrome (CFS). We examined the sensitivity of theimmune system to the glucocorticoid agonist dexamethasone and the􏰁2-adrenergic agonist terbutaline in 15 adolescent girls with CFS and14 age- and sex-matched controls. Dexamethasone inhibits T-cellproliferation in healthy controls and in CFS patients. However, themaximal effect of dexamethasone on T-cell proliferation is signifi-cantly reduced in CFS patients as compared with controls. The 􏰁2-adrenergic receptor agonist terbutaline inhibits tumor necrosis fac-tor-􏰀 production and enhances interleukin-10 production by

CHRONIC fatigue syndrome (CFS) is a disease char-acterized by debilitating fatigue for at least 6 monthsthat has resulted in a substantial reduction in the activitylevel of the individual and is not attributable to knownclinical conditions. For study purposes, the Centers forDisease Control and Prevention (Atlanta, GA) have de-fined criteria for CFS (1). In addition to the persistent orrelapsing fatigue, at least four other symptoms from a listof eight should be present concurrently with the fatigue.These symptoms include unrefreshing sleep, postexertionmalaise, multi-joint pain, new headaches, muscle pain,tender cervical or axillary lymph nodes, sore throat, andimpaired memory or concentration (1).

The etiology of CFS is unknown. Viral infections have beensuggested as precipitating events, and a number of studiessuggest involvement of viruses in at least part of the patients(2– 4). In a recent study on children and adolescents with CFS,60% of the patients indicated an acute disease at onset (5).However, to date, there is no evidence for a specific virusassociated with CFS (6).

Other studies have focused on immunological dysfunctionin CFS patients and suggested changes in cytokine produc-tion, natural killer cell activity, and alterations in T-cell re-activity (7–9). Although immunological changes have been

Received August 25, 1999. Revision received October 21, 1999. Ac-cepted October 25, 1999.

Address correspondence and requests for reprints to: Dr. AnnemiekeKavelaars, Wilhelmina Children’s Hospital of the University MedicalCenter Utrecht, Department of Pediatric Immunology, Room KC03.068.0, Lundlaan 6, 3584 EA Utrecht, The Netherlands. E-mail:a.kavelaars@wkz.azu.nl.

* Supported by a grant of the “ME-fonds” of The Netherlands.

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monocytes. Our data demonstrate that the capacity of a 􏰁2-adrenergicagonist to regulate the production of these two cytokines is also re-duced in CFS patients. We did not observe differences in baseline orCRH-induced cortisol and ACTH between CFS patients and controls.Baseline noradrenaline was similar in CFS and controls, whereasbaseline adrenaline levels were significantly higher in CFS patients.

We conclude that CFS is accompanied by a relative resistance of theimmune system to regulation by the neuroendocrine system. Based onthese data, we suggest CFS should be viewed as a disease of deficientneuroendocrine-immune communication. (J Clin Endocrinol Metab85: 692– 696, 2000)

described, a consistent pattern of immunological abnormal-ities has not been found.

In adults with CFS, evidence has been presented forchanges in the neuroendocrine system. Demitrack et al. (10)showed that the reactivity of the hypothalamo-pituitary-ad-renal (HPA) axis is decreased in adult patients with CFS ascompared with controls. Unfortunately, treatment with hy-drocortisone results in only limited improvement in CFSpatients. Cleare et al. (11) presented data showing that oraladministration of low doses of hydrocortisone could im-prove the clinical condition in about 30% of a selected groupof patients. In another study it was shown that oral hydro-cortisone administration did result in some improvement asmeasured by a change in Wellness score, which is a globalhealth scale. However, fatigue and activity did not changesignificantly in this study (11).

In view of the generalized symptomatology in CFS, thesearch for a single factor cause does not seem to be the mostadequate approach. We hypothesized that the symptomatol-ogy in CFS may result from abnormalities in interorgan com-munication rather than from abnormalities in a single organsystem. One aspect of interorgan communication is based onthe production and secretion of (neuro)endocrine mediatorsby a given organ system and the presence and reactivity ofspecific receptors in the target organ system(s). Thus, notonly alterations in the actual level of neuroendocrine medi-ators, but also changes in the way target organs respond tothese mediators, may result in inadequate communication.Inadequate communication could contribute to the patho-physiology in CFS and may explain the mixed results ob-served in various studies that focus on a single organ system.

It is now well established that the neuroendocrine system

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and the immune system closely interact. Psychological stresscan modulate immune reactivity via complex interactionsinvolving the HPA axis as well as the autonomic nervoussystem (12–14). Cells of the immune system, like cells in otherorgan systems, express receptors for hormones and neuro-transmitters (14, 15). Triggering of these receptors results inmodulation of immune reactivity. As a model system toinvestigate the integrity of neuroendocrine regulation wechose cells of the immune system that are easily accessible inthe peripheral blood and can be studied ex vivo.

We determined the sensitivity of the immune system toregulation by the glucocorticoid agonist dexamethasone andthe 􏰁2-adrenergic receptor agonist terbutaline. It has beenwell established that glucocorticoid receptor agonists willinhibit the proliferative response of T cells (16–18). There-fore, we determined the effect of dexamethasone on T-cellproliferation in healthy individuals and in CFS patients. 􏰁2-adrenergic receptor agonists are known to regulate cytokineproduction by monocytes (19–21). Thus, we examinedchanges in lipopolysaccharide (LPS)-induced production ofthe cytokines tumor necrosis factor (TNF)-􏰀 and interleukin(IL)-10 in the presence of increasing concentrations of ter-butaline. We also examined baseline levels of epinephrineand norepinephrine in the same blood samples. In addition,plasma cortisol and ACTH levels before and after infusion ofCRF were determined.

Patients and Methods

Patients

Fifteen girls with CFS, according to the criteria defined by the Centersfor Disease Control and Prevention, with a substantial decrease in ac-tivity level and no primary psychological morbidity were asked to enterour study. The CFS patients included in our study were not taking anymedication at the time of the study or within 6 weeks before the study.Patients with a psychiatric history were excluded. The body mass indexof patients was significantly higher in CFS patients than in controls.Control individuals were recruited from healthy schoolmates of thepatients of similar age and the same sex. An iv line was inserted into theunderarm between 0830 and 0900 h. After a 60-min rest, a blood samplewas drawn for analysis of plasma catecholamines and for determinationof receptor sensitivity. The CRH infusion was done between 1300 and1400 h. The experimental protocol was approved by the medical ethicalcommittee of the Wilhelmina Children Hospital. Written informed con-sent was obtained from parents and from the children.

Ex vivo response of peripheral blood cells to dexamethasone

Whole blood was diluted 1:10 in medium [RPMI 1640 (Gibco, GrandIsland, NY) supplemented with 2 mm glutamine, 100 U/mL penicillin,and 100 􏰂g/mL streptomycin]. Diluted blood (100 􏰂L) was cultured for96 h in round bottom 96-well plates (Nunc, Glostrup, Denmark) with 25􏰂L phytohemagglutinin (PHA) (HA 15; Murex Diagnostics, Dartford,UK), final concentration 25 􏰂g/mL, and 25 􏰂L DEX in the concentrationsindicated. At 16–18 h before the end of the culture, 1 􏰂Ci (37 kBq)[3H]-thymidine was added. At the end of the culture period, cells wereharvested by the use of an automated cell harvester, and incorporatedradioactivity was determined in a liquid scintillation counter.

Ex vivo response of peripheral blood cells to terbutaline

Whole blood was diluted 1:10 in medium, and 100 􏰂L diluted bloodwas cultured with 50 􏰂l LPS [Escherichia coli (DIFCO Laboratories, De-troit, MI); final concentration 2 ng/mL] and 50 􏰂L medium or the􏰁2-adrenergic receptor agonist terbutaline (Sigma Chemical Co., St.Louis, MO). After 18 h of culture at 37 C, supernatants were harvestedand stored at 􏰄80 C until analysis. TNF-􏰀 and IL-10 levels were deter-

mined by enzyme-linked immuosorbent assay (Pelikine; CLB, Amster-dam, The Netherlands).

Determination of plasma catecholamines

Two milliliters of blood were collected on ice in 0.25 mol/L EGTA and0.2 mol/L glutathione. Plasma samples were stored at 􏰄80 C. Cat-echolamines were determined by high-performance liquid chromatog-raphy according to the method described by Willemsen et al. (22). Thedetection limits were: adrenaline, 2 pg/mL; and noradrenaline, 2 pg/ml;CV, 􏰃10%.

CRH induced changes in ACTH and cortisol

CRH (100 􏰂g) was infused via an iv line between 1300 and 1400 h.Before and at various time points after infusion of CRH, blood wascollected in ethylenediaminetetraacetate-coated tubes on ice.

Plasma cortisol was determined using a fluorescence polarizationimmunoassay (Abbott Laboratories, Abbott Park, IL) (detection limit,0.64 􏰂g.dL; CV, 􏰃5%).

Plasma ACTH was determined by RIA, using antiserum from IgGCorporation USA and 125I-ACTH from CIS Bioindustries (France) (de-tection limit, 20 ng/L; CV, 􏰃8%).

Data analysis

Dose-response curves were analyzed by nonlinear regression usingGraphPad Software, Inc. Prism 3.0 software. Data are expressed as meanand sem. Two-tailed Student’s t tests were used to compare groupdifferences. P 􏰃 0.05 was considered statistically significant.

Results

Subject characteristics

We examined 15 patients diagnosed with CFS and 14healthy controls. The mean age in the patient group was15.8 􏰆 0.4 yr (range, 11–17) and in the control group 14.5 􏰆0.6 yr (range, 10–17). Mean duration of disease was 21.8 􏰆3.9 months (range, 6 – 48).

Ex vivo response of peripheral blood cells to dexamethasone

Whole blood cultures were stimulated with the T cell mi-togen PHA to induce proliferation and increasing concen-trations of the glucocorticoid agonist dexamethasone. In theabsence of dexamethasone, proliferative responses in CFSpatients were higher than in healthy controls (CFS, 45,170 􏰆5,063 cpm, n 􏰅 15; controls, 31,700 􏰆 3,852, n 􏰅 14; P 􏰅 0.044).

As expected, The addition of dexamethasone to the cul-tures resulted in a dose-dependent inhibition of the prolif-erative response (Fig. 1). Interestingly, however, the effect ofdexamethasone is much less pronounced in cultures withcells from CFS patients (Fig. 1). The maximal effect of dexa-methasone was 88.1 􏰆 3.1% inhibition of T-cell proliferationin healthy controls. In contrast, in CFS patients the maximaleffect was 66 􏰆 4.9% inhibition, which is significantly lower(P 􏰅 0.001). The IC50 was similar in CFS patients and controls(CFS, 31 nm; controls, 47 nm).

Ex vivo response of peripheral blood cells to terbutaline

To examine the sensitivity of the immune system to􏰁2-adrenergic regulation, we investigated the effect of the􏰁2-adrenergic receptor agonist terbutaline on cytokineproduction by peripheral blood cells. Whole blood cul-tures were stimulated with LPA for 18 h to induce mono-

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694 KAVELAARS ET AL.

FIG. 1. DexamethasoneinhibitionofT-cellproliferation.Wholebloodcultures were stimulated with PHA (25 􏰂g/mL) in the presence ofincreasing concentrations of dexamethasone. After 72 h, cultureswere pulsed with 1 􏰂Ci 3H-thymidine. Cells were harvested after 96 hof culture, and incorporation of 3H-thymidine was determined as ameasure of T-cell proliferation. Data are expressed as percentage ofproliferation in the absence of dexamethasone and represent themean and SEM. E, controls, n 􏰅 14; F, CFS, n 􏰅 15.

cyte cytokine production, and increasing concentrations ofterbutaline were added to the cultures.

In the absence of terbutaline, TNF-􏰀 production did notdiffer significantly between patients and controls (CFS,473.9 􏰆 104.6 pg/mL, n 􏰅 15; controls, 799.1 􏰆 205.1 pg/mL,n 􏰅 14; P 􏰅 0.17). The data depicted in Fig. 2 clearly dem-onstrate that the addition of the 􏰁2-adrenergic agonist resultsin inhibition of TNF-􏰀 production. More importantly, ourdata demonstrate that the inhibitory effect of the 􏰁2-adren-ergic agonist on TNF-􏰀 production is significantly lower inCFS patients than in controls. In control subjects, the maximaleffect of terbutaline on TNF-􏰀 production was 67 􏰆 1.3%inhibition. In CFS patients, maximal inhibition of TNF-􏰀production was only 37.1 􏰆 3.3% (P 􏰃 0.0001). There was nodifference in IC50 between CFS and controls (CFS, 3.9 nm;controls, 8.2 nm).

􏰁2-adrenergic receptor agonists inhibit TNF-􏰀 production,but enhance IL-10 production. If the decreased inhibition ofTNF-􏰀 production in CFS patients is the result of alterationsin 􏰁2-adrenergic receptor function, then we expect a smaller􏰁2-adrenergic agonist-induced increase in IL-10 productionin CFS patients, as well. The data in Fig. 2 demonstrate thatthe 􏰁2-adrenergic agonist is less capable of increasing IL-10production in CFS patients than in controls (maximal in-crease: CFS, 45 􏰆 6.3; controls, 70 􏰆 5.8%; P 􏰅 0.007). In theabsence of terbutaline, there was no difference in IL-10 pro-duction (CFS, 64.1 􏰆 11.4 pg/mL, n 􏰅 14; controls, 44.3 􏰆 8.7pg/mL, n 􏰅 13; P 􏰅 0.18).

Plasma adrenaline and noradrenaline

Plasma noradrenaline levels in CFS patients did not differfrom levels in healthy subjects (CFS, 1.47 􏰆 0.1 nmol/L, n 􏰅14; control, 1.46 􏰆 0.2 nmol/L, n 􏰅 14; P 􏰅 0.98). There wasa statistically significant increase in plasma adrenaline levelsin CFS patients as compared with controls (CFS, 0.14 􏰆 0.03nmol/L, n 􏰅 14; control, 0.07 􏰆 0.01 nmol/L, n 􏰅 14; P 􏰅0.04).

Reactivity of the pituitary-adrenal axis

At baseline, plasma ACTH and cortisol levels were similarin patients and controls. Plasma cortisol levels were similarin patients and controls (cortisol: CFS, 0.28 􏰆 0.03 􏰂mol/L,n 􏰅 15; control, 0.29 􏰆 0.03 􏰂mol/L, n 􏰅 14; P 􏰅 0.85. ACTH:CFS, 42.7 􏰆 4.8 ng/L, n 􏰅 15; control, 35.4 􏰆 3 ng/L, n 􏰅 14;P 􏰅 0.21). The data in Fig. 3 show that the CRH-inducedincrease in plasma ACTH and plasma cortisol is also similarin CFS patients and controls.

Discussion

The pathophysiology of CFS is poorly understood. Re-search over the past 10 yr indicates that the syndrome cannotbe explained by defects in one single organ system. Thepresent study did not aim at defining alterations in one or theother system, but was designed to investigate the integrity ofinterorgan communication in CFS patients.

As a model system for interorgan communication, wechose the interaction between neuroendocrine factors andthe immune system. The reactivity of the immune system canbe tested ex vivo, and modulatory effects of glucocorticoids,as well as of 􏰁2-adrenergic receptor agonists, have beenclearly defined. Our results demonstrate that in adolescents

With CFS, communication between neuroendocrine systemand immune system is altered. In ex vivo studies, usingperipheral blood of CFS patients, we demonstrated that thesensitivity of the immune system to regulation by neuroen-docrine factors is decreased. T-cell proliferation is less sen-sitive to the inhibitory effects of dexamethasone, and mono-cyte cytokine production is relatively resistant to themodulatory effects of a 􏰁2-adrenergic receptor agonist.

Interestingly, the decreased sensitivity to GC and to a􏰁2-adrenergic receptor agonist becomes apparent as a de-creased maximal effect rather than as a change in the EC50.These results suggest that either the number of functionalreceptors is reduced or that the transduction of the signalfrom the receptor to the intracellular effector system is di-minished in CFS. A reduced number of functional receptorsis often associated with high plasma levels of the hormone.In that case, the receptor is already occupied by hormone invivo, and a lower number of receptors is available for exog-enous ligand added ex vivo. However, in our study group,we have no evidence for disturbances in plasma cortisol thatcould explain the relative resistance to dexamethasone of Tcells from CFS patients on this level. Baseline cortisol andCRH-induced increases in cortisol were similar in CFS pa-tients and healthy subjects. Moreover, plasma ACTH levelsbefore and after CRH infusion are similar in CFS and con-trols. Therefore, we conclude that there are no major abnor-malities in the reactivity of the HPA-axis in adolescents with

CFS. In line with our data, baseline cortisol and ACTH inadults with CFS were not significantly different from con-trols in a number of studies (23). Demitrack et al. (10) alsoreported normal baseline cortisol levels, however, decreased24-h excretion of cortisol in urine and a blunted response toinfusion with CRH in a group of adults with CFS. It ispossible that we do not observe changes in HPA-axis reac-tivity in our group of CFS patients because we are studyinga much younger population. In our group of adolescentswith CFS, mean age was 15.8 yr, whereas Demitrack et al. (10)studied adults with a mean age of 36.9 yr. Moreover, meanduration of disease in the adult study was 7.2 yr, whereas inour patient group mean duration of disease was less than 2yr (10).

Our present data showing that cells of CFS patients arerelatively resistant to a glucocorticoid receptor agonist maybe of interest in view of the limited effect of hydrocortisonetreatment in patients with CFS who do have a reduced ac-tivity of the HPA-axis (11, 24). If the resistance to GC agonistin CFS is a more generalized phenomenon, then normaliza-tion of plasma GC levels may not be sufficient to restorecommunication.

Alterations in neuroendocrine-immune communicationare not specific for CFS. In a previous study, we demon-strated that the maximal effect of a 􏰁2-adrenergic agonist onTNF-􏰀 production is increased in patients with rheumatoidarthritis (25). Interestingly, peripheral blood cells of rheu-matoid arthritis patients are more sensitive to the 􏰁2-adren-ergic receptor agonist, whereas in CFS we observe decreasedreactivity. In addition, in both diseases we did not observealterations in EC50, but only changes on the level of themaximal effect of the agonist (25). The increased sensitivityof peripheral blood cells of rheumatoid arthritis patients to􏰁2-adrenergic modulation seemed to be associated with de-creased expression of GRK-2, an intracellular kinase thatplays a major role in receptor desensitization (25). Thus,altered sensitivity to a 􏰁2-adrenergic agonist can be due to achange in the coupling efficiency of the receptor, a processin which GRK-2 plays a crucial role (26). In animal modelsit has been shown that chronic infusion of a 􏰁2-adrenergicagonist will result in increased levels of GRK-2 and concom-itantly in relative resistance to regulation by 􏰁2-adrenergicagonists (27, 28). It is conceivable that in CFS the decreasedreactivity of the immune system to a 􏰁2-adrenergic agonistis associated with increased levels of this kinase since plasmaadrenaline levels are increased in CFS patients.

The question remains how the altered sensitivity of theimmune system for neuroendocrine regulation developed.Was it a preexisting condition or is it the result of the disease?It may well be possible that the altered neuroendocrine-immune communication is associated with a high level ofpsychological stress. We know that the psychological stressof bereavement results in a significant decrease in the sen-sitivity of the immune system to dexamethasone (manuscriptin preparation). A high level of psychological distress has notonly been reported in adults, but also in adolescents with CFS(29–31). Interestingly, a large study on postinfection fatigueshows that there is an association between psychologicaldistress and fatigue prior to viral infection and the likelihoodto develop chronic fatigue later on (32). We hypothesize that

NEUROENDOCRINE-IMMUNE INTERACTION IN CFS 695

The abnormalities in neuroendocrine-immune communication in chronic fatigue syndrome result from the level ofpreexisting psychological distress and a precipitating event(e.g. a viral infection).

In summary, the present study demonstrates that the in-teraction between neuroendocrine mediators and a targetsystem, the immune system, is disturbed in CFS. Additionalstudies should be performed to get insight in the role of theseabnormalities in the pathophsyiology of CFS.

Acknowledgments

We gratefully acknowledge Marijke Tersteeg and Jitske Zijlstra forexcellent technical assistance.

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