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Stress-induced changes in LPS-induced pro-inflammatory cytokine production in chronic fatigue syndrome
Psychoneuroendocrinology (2005) 30, 188–198
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Stress-induced changes in LPS-induced pro-inflammatory cytokine production in chronic fatigue syndrome Jens Gaab*, Nicolas Rohleder1, Vera Heitz, Veronika Engert, ¨rmeyer, Ulrike Ehlert2 Tanja Schad2, Thomas H. Schu Center for Psychobiological and Psychosomatic Research, University of Trier, Trier, Germany Received 18 March 2004; received in revised form 11 June 2004; accepted 12 June 2004
KEYWORDS Chronic fatigue syndrome; HPA axis; Psychosocial stress test; Pro-inflammatory cytokines; Il-6; TNF-a; ACTH; Cortisol; LPS
Summary Objective. It has been suggested that a hypofunctional hypothalamic– pituitary–adrenal (HPA) axis in chronic fatigue syndrome could result in an exaggerated release of pro-inflammatory cytokines during stress. As pro-inflammatory cytokines are involved in the induction of sickness behavior and thus constitute a potential physiological correlate of stress-induced symptom exacerbation in chronic fatigue syndrome, we set out to evaluate the LPS-induced production of proinflammatory cytokines during psychosocial stress in CFS and healthy controls. Method. Twenty-one CFS patients and 20 healthy controls matched for age and gender underwent a standardized psychosocial stress test (Trier social stress test, TSST). Adrenocorticotropine hormone (ACTH), salivary cortisol and plasma cortisol levels were measured before and repeatedly following exposure to the stressor. Lipopolysaccharide-stimulated production of interleukin-6 and tumor necrosis factor-alpha were assessed at baseline as well as 10 and 60 min after the stress test. Results. CFS patients showed an inverse stress-induced response pattern of LPSstimulated cytokines responses in comparison to healthy controls, i.e. stimulated cytokine production decreased shortly after stress in CFS patients, while it increased in controls. Fatigue scores and basal LPS-induced cytokine levels were significantly associated for TNF-a in controls and for both cytokines in CFS patients. Stressinduced changes in stimulated cytokine production were not associated with general fatigue scores in the control group, whereas in the CFS group, fatigue scores were significantly correlated with integrated levels of LPS-induced cytokines.
Abbreviations: CFS, chronic fatigue syndrome; ACTH, adrenocorticotropine hormone; PC, plasma cortisol; SC, saliva cortisol; HR, heart rate; TSST, Trier social stress test; CLIA, chemiluminscence assay; SIP, sickness impact profile; HADS, hospital anxiety and depression scale; SCL-90R, symptom checklist; AUC, area under curve. * Corresponding author. Present address: Institute for Psychology, Clinical Psychology and Psychotherapy, University of Zu ¨rich, Zu ¨richbergstr. 43, Zu ¨rich, Switzerland. Tel.: C41-1-6343096; fax: C41-1-6343696. E-mail address: [email protected] (J. Gaab). 1 Present address: Department of Biopsychology, Technical University of Dresden, Dresden, Germany. 2 Present address: Institute for Psychology, Clinical Psychology and Psychotherapy, University of Zu ¨rich, Zu ¨richbergstr. 43, Zu ¨rich, Switzerland. 0306-4530/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.
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However, partial correlations revealed that these results were due to the high correlations with basal LPS-induced cytokine levels. Conclusion. CFS patients do not show an exaggerated secretion of LPS-induced cytokines. Although cortisol responses to stress were normal, pro-inflammatory cytokine levels in CFS patients were significantly attenuated. Possible intracellular mechanisms, such as for example an enhanced sensitivity to inhibitory effects of glucocorticoids, a diminished responsivity to catecholaminergic stimulation, and a disruption of intracellular activation are discussed. Basal levels of stimulated proinflammatory Il-6 levels are generally related to fatigue scores. However, in CFS patients this association is of greater magnitude and can also be observed for TNF-a. Q 2004 Elsevier Ltd. All rights reserved.
1. Introduction Chronic fatigue syndrome (CFS) is characterized by persistent or relapsing debilitating mental and physical fatigue, which is exacerbated by minor exertion. Patients also experience further somatic symptoms, such as myalgia, arthralgia, cognitive disturbances, low-grade fever, and sleep disturbances (Fukuda et al., 1994). Although these symptoms are indicative of a somatic origin, and despite a large number of empirical studies, no objective and specific somatic pathology has been identified. Consequently, the diagnosis of CFS is established by the exclusion of somatic and psychiatric causes of chronic fatigue. However, dysregulations of somatic systems involved in the maintenance of homeostasis have been observed and proposed as physiological substrates of symptoms experienced (Natelson, 2001). Amongst other candidates, the interaction of psychological, endocrine, and immune processes seems to be of particular relevance, since on the one hand psychological and behavioral factors associated with the precipitation and perpetuation of the syndrome, such as stress, inactivity and sleep problems (Luger et al., 1987; Leese et al., 1996; Theorell et al., 1999), are also known to influence endocrine and consequently immune functioning (for review: (McEwen et al., 1997) and on the other hand, alterations of neuroendocrine and immune mechanisms can be linked to CFS symptoms (for review: (Clauw and Chrousos, 1997; Lariviere and Melzack, 2000; Dantzer, 2001). From this perspective, alterations of the bidirectional communication between the hypothalamic–pituitary–adrenal axis and the immune system or, more precisely, between glucocorticoids and cytokines have been discussed as a possible etiological model for CFS (for review: (VollmerConna et al., 1998; Patarca, 2001). In general, glucocorticoids modulate the immune response by inhibiting the production and release of
pro-inflammatory cytokines, thus promoting the switch from a T helper (Th) 1- to Th 2-dependent or cellular to humoral immune response respectively. With regard to CFS, it is noteworthy that proinflammatory cytokines are mediators of the behavioral and motivational changes seen during infections, referred to as ’sickness behavior’ (Dantzer, 2001), and elevated levels of or treatment with pro-inflammatory cytokines have been shown to be associated with fatigue symptomatology (Papanicolaou et al., 1996b; Vgontzas et al., 2002; Cleeland et al., 2003). Since pro-inflammatory cytokines are released during psychosocial and physiological stress (Papanicolaou et al., 1996a; Goebel et al., 2000; Steptoe et al., 2001), a hypersecretion of pro-inflammatory cytokines during stress due to a hypofunctional neuroendocrine counterregulation could serve as a possible explanation of exercise- and stress-induced exacerbation of fatigue experienced by patients with CFS (Wood et al., 1994; Lutgendorf et al., 1995). Several studies have investigated dysregulations of the HPA axis (for review: (Cleare, 2003) and alterations of circulating cytokine levels or stimulated cytokine production in CFS (for review: (Visser et al., 2000; Patarca, 2001). However, findings of HPA axis dysregulations and alterations of cytokine levels and production are heterogeneous, possibly reflecting both the heterogeneity of the subjects under study with regard to duration of symptoms, inactivity, sleep disturbance, psychiatric comorbidity, medication, and ongoing stress as well as the heterogeneity of methods and study designs used. With regard to the interaction of neuroendocrine parameters and pro-inflammatory cytokine production in CFS, Visser and colleagues reported supersensitivity of lipopolysaccharide (LPS) -induced IL-10 and IFN-g, but not of IL-12 or IL-4 production to inhibition by dexamethasone, independent of the number or affinity of glucocorticoid receptors (GR) (Visser et al., 1998, 2001a,b).
190 A number of studies have assessed immune responses during or after stress in CFS. Cannon et al. (Cannon et al., 1998) reported a disrupted endocrine influence on neutrophil mobilization in CFS patients undergoing a standardized exercise program, without indication of an excessive immune response. Other studies employing mild exercise tests were unable to find significant differences in cytokine levels between CFS patients and controls (Lloyd et al., 1994; Peterson et al., 1994; LaManca et al., 1999). In contrast to these studies using physical exercise tests, recent reports suggest an enhanced sensitivity of circulating leucocytes to increasing doses of dexamethasone in CFS patients undergoing a standardized psychosocial stress paradigm (Gaab et al., 2003c). In summary, the results of previous studies on this topic are either conflicting or do not support the assumption on an attenuated suppression of pro-inflammatory cytokine responses by glucocorticoids. However, the hypothesis that an altered neuroendocrine-immune interplay during stress could be used to explain fatigue symptoms has not been tested directly as yet. We therefore set out to assess the LPS-induced production of pro-inflammatory cytokines before and after a standardized psychosocial stress test in CFS patients and healthy controls and related these findings to HPA axis responses and general fatigue symptoms.
J. Gaab et al. and no medical cause for the chronic fatigue in routine laboratory testing. All patients were medically examined by the same physician (THS), according to recommendation (Fukuda et al., 1994), and were also interviewed by a trained psychologist (JG). This consisted of a computeraided standardized and structured diagnostic interview in accordance with the Diagnostic and Statistical Manual of Mental Health Disorders 4th Edition (Wittchen and Pfister, 1997) and a semistructured CFS interview, which was concerned with the severity and course of all symptoms required by the US and UK definitions (Sharpe et al., 1991; Fukuda et al., 1994). All patients fulfilled US and UK consensus criteria for the diagnosis of CFS (Sharpe et al., 1991; Fukuda et al., 1994). Patients were matched for age and gender with healthy volunteer controls, randomly recruited via telephone calls. Controls were free of medication and underwent comprehensive medical examination for past and current health problems. Control subjects were screened for any current or lifetime psychiatric symptoms or disorders by a clinical psychologist (JG). After subjects were provided with complete written and oral descriptions of the study, written informed consent was obtained. The study sample is part of a larger CFS patient cohort, the results of which have been published elsewhere (Gaab et al., 2002a,b).
2.2. Procedure
2. Methods 2.1. Subjects The study was approved by the Ethics Committee of the Medical Council of Rheinland-Pfalz, Germany. Patients were contacted through a German selfhelp organization. Interested parties received a postal screening questionnaire, containing all symptoms required by the UK and US definitions of CFS (Sharpe et al., 1991; Fukuda et al., 1994). Patients fulfilling the symptom requirements in the screening questionnaire were interviewed over the telephone and asked to disclose any diagnosed medical illnesses and psychiatric disorders. Prospective participants were only excluded from the study if they had received a medical or psychiatric diagnosis defined as an exclusion criterion by the US definition (Fukuda et al., 1994). Selection criteria for participation in the study were fulfillment of CFS symptom criteria in the postal screening questionnaire, new or definite onset of CFS, age between 30–50, no current antidepressant, anxiolytic, antibiotic, antihypertensive, and steroid medication
2.2.1. Trier social stress test (TSST) The TSST has been repeatedly found to induce profound endocrine and cardiovascular responses in 70–80% of the subjects tested (Kirschbaum et al., 1993). After basal blood and saliva samples were taken, subjects were introduced to the TSST (1 min). They were given 3 min to prepare themselves for a fake job interview (5 min), followed by a mental arithmetic task in front of an audience (5 min). Subjects were told that they would be videotaped for further analysis of their behavior. Immediately after the test, a blood and saliva sample was taken, with further blood and saliva samples taken at 10, 20, 30, 45, and 60 min to assess endocrine parameters. Blood was drawn into 9 ml ethylenediamine tetraacetate (EDTA) monovettes (Sarstedt, Nu ¨mbrecht, Germany). Additional samples were taken immediately before as well as 10 and 60 min after TSST for stimulation of inflammatory cytokine production into heparinized sterile syringes (Braun, Melsungen, Germany) and for a differential blood count into 2.3 ml EDTA monovettes (Sarstedt, Nu ¨mbrecht, Germany).
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2.3. Measures 2.3.1. Sampling methods and biochemical analyses EDTA blood samples for endocrine measures were spun immediately at 4 8C and stored at K20 8C until assayed. Saliva was collected by the subjects themselves using Salivette collection devices (Sarstedt, Nu ¨mbrecht, Germany) and stored at room temperature until completion of the session. It was then stored at K20 8C until biochemical analysis was carried out. ACTH and PC were measured with two-site commercial chemiluminescence assays (CLIA, Nichols Institute Diagnostics, Bad Nauheim, Germany). The free cortisol concentration in saliva (SC) was determined using a time-resolved immunoassay with fluorometric detection, as described in detail elsewhere (Dressendorfer et al., 1992). Inter- and intraassay coefficients of variance were below 10% for all analytes. For the assessment of inflammatory cytokines, whole blood was coincubated with LPS (E. Coli, Difco, Augsburg, Germany) on a 24-well plate (Greiner, Nuertingen, Germany). Diluted whole blood (400 ml) was added to 50 ml of LPS. The final concentration was 30 ng/ml LPS. After 18 h of incubation at 37 8C and 5% CO2, the plates were centrifuged for 10 min at 2000 g at 4 8C. The plasma supernatant was collected and stored at K80 8C until assayed. IL-6 and TNF-alpha were measured using ELISA, employing the multiple antibody sandwich principle (BD Pharmingen, San Diego, CA, USA). Plates were read by a microplate reader (Anthos HTII, Anthos Labtec, Salzburg, Austria), and absorbance was transformed to cytokine concentration (pg/ml) using a standard curve computed by Anthos Winread 2.3 software (Anthos Labtec, Salzburg, Austria). Inter- and intraassay coefficients of variation were below 10%. To correct for changing numbers of monocytes, cytokine levels are expressed as ng relative to one million monocytes. A differential blood count was performed, measuring absolute numbers of leukocytes, as well as number and percentage of monocytes, lymphocytes, and granulocytes. Cell counting was performed on a Coulter AcTdiff cell counter (BeckmanCoulter, Krefeld, Germany). 2.3.2. Psychometric measures and patient characteristics To assess the severity of the most prominent CFS symptoms, all subjects completed a German translation of the fatigue scale (FS) (Chalder et al., 1993). To be able to assess the general fatigue
191 severity, the FS was administered at least one day before the psychosocial stress test. The FS is an 11-item self-report measure developed to assess fatigue. It consists of two scales, which assess physical and mental fatigue. We used a 0, 1, 2, 3 scoring system and calculated a total score. An internal consistency (Cronbach’s alpha) of aZ0.96 for the total score has been calculated on a larger CFS population (NZ193) (Gaab et al., unpublished data). In addition, all subjects completed a battery of questionnaires, including the sickness impact profile (SIP) (Kessler et al., 1997), the hospital anxiety and depression scale (HADS), which measures symptoms occurring during the last week (Herrmann et al., 1995), and the symptom checklist (SCL-90R), which assesses symptoms experienced during the last four weeks (Franke, 1995). All of these scales have been evaluated on German patient populations, with good validity and reliability. Duration of CFS was assessed in months since onset of symptoms.
2.4. Statistical analysis c2 analysis was used to test for significant differences in discrete variables. ANOVAS were computed to analyze endocrine and immune parameters between groups. Correlations were computed as Pearson product-moment correlations and partial correlations. For all endocrine parameters, areas under the total response curve (AUC), expressed as area under all samples, were calculated using the trapezoidal method (Pruessner et al., 2003). To test for possible differences between correlations, coefficients were Fisher-Z transformed and tested for significance using z-distribution tables. KolmogorovSmirnov tests revealed that immune data was not normally distributed. Calculating the log of cytokine values produced nearly normally distributed values. Although log-transformed cytokine values were used for all statistical analyses, means and standard deviations of untransformed values are presented. Data was also tested for homogeneity of variance using Levene’s test before statistical procedures were applied. For all analyses, significance levels were aZ5%. Unless indicated, all results shown are meansGstandard error of means (SEM).
3. Results 3.1. Sample characteristics Twenty-one CFS patients and 20 controls participated in the study. Gender ratio, number of
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Table 1
Demographic and psychometric characteristics of CFS patients and healthy controls.
Sex (male/female) TSST Age in yearsa BMIa Fatigue scale HADSb Depression Anxiety SCL-90Rb Anxiety Phobic anxiety Depression Somatization SIPb Home management Ambulation Mobility Alertness behavior Sleep and rest Social interaction a b
CFS
Controls
10/11 36.00 (30–47) 22.4 (17.6–26.8) 25.76 (0.95)
11/9 35.24 (29–44) 24.1 (18.1–34.6) 9.45 (0.98)
7.3 (0.9) 6.4 (0.9)
1.4 (0.3) 3.2 (0.5)
58.4 53.2 59.7 70.4
45.3 (1.5) 47 (1.5) 44.2 (1.8) 44.8 (1.6)
(2.2) (2.8) (2.2) (2.5)
13.5 (2.5) 6.0 (1.5) 7.1 (2.0) 33.9 (4.4) 27.6 (4.2) 16.8 (2.4)
0.00 0.00 0.25 0.00 0.00 0.73
(0.0) (0.0) (0.2) (0.0) (0.0) (0.4)
c2Z0.22; PZ0.64 t(44)ZK0.56; PZ0.57 t(44)Z1.63; PZ0.11 F (1/39)Z143.5; P!0.001 F (2/43)Z24.6; P!0.001 F (1/44)Z43.7; P!0.001 F (1/44)Z9.4; PZ0.004 F (9/36)Z12.45, P!0.001 F (1/44)Z27.5; P!0.001 F (1/44)Z3.45, PZ0.07 F (1/44)Z30.8, P!0.001 F (1/44)Z55.0, P!0.001 F (6/36)Z12.81, P!0.001 F (1/41)Z30.5, P!0.001 F (1/41)Z17.7, P!0.001 F (1/41)Z11.5, P!0.001 F (1/41)Z63.2, P!0.001 F (1/41)Z33.7, P!0.001 F (1/41)Z46.8, P!0.001
Mean (range). Mean (SEM).
subjects, mean age, and body mass index (BMI) did not differ significantly between the groups (Table 1). The mean duration of patients’ symptoms was 64.0 months, with a range from 17 to 168 months. Fourteen CFS patients reported an infectious onset of their symptoms. All patients reported an onset of symptoms within three months. The FS, HADS, SCL-90R and SIP scores of the CFS group were significantly higher than those of the control group and comparable to reported scores in previous studies (Table 1). One CFS patient fulfilled the criteria for a current Episode of Major Depression. However, since the exclusion of this subject did not alter the results of the analysis, the patient was included in the reported analysis. Seven patients reported a past history of major depression and four reported a past history of anxiety disorder. None of the controls reported any current or lifetime psychiatric disorder.
3.2. HPA axis responses in the TSST The TSST caused significant endocrine responses over time (ACTH: F (6/234)Z45.54; P!0.001; PC: F (6/234)Z39.57; P!0.001; SC: F (6/234)Z26.29; P!0.001). ANOVA revealed significant response differences over time between the groups for ACTH, but not for any cortisol parameter in the TSST (ACTH: F (1.69/65.9)Z3.47; PZ0.04; PC: F (2.59/101.06)Z1.19; PZ0.31; SC: F (2.5/99.74)Z
0.42; PZ0.70; Fig. 1). In addition, the area under the ACTH response curve was significantly reduced in patients compared to controls (F (1/39)Z6.34; PZ0.02;), but no differences in the cortisol parameters were observed (PC: F (1/39)Z0.1; PZ0.91; SC: F (1/39)Z1.03; PZ0.32; Fig. 1).
3.3. Immune responses in the TSST The total number of leukocytes increased significantly following psychosocial stress in both groups (time effect: F(1.36/46.24)Z3.923, PZ0.042). However, leukocyte numbers did not differ between patients and controls over time (group by time interaction effect: F(1.36/46.24)Z0.22, PZ 0.72; see Table 2, upper row). No stress-related changes in monocyte numbers or any group differences were observed (group: F(1/39)Z0.08, PZ 0.79; group by time interaction: F(1.54/52.61)Z 1.66, PZ0.20; see Table 2, lower row). The TSST caused significant group-specific responses in stimulated cytokine production (group by time interaction: Il-6: F (1.66/64.65)Z 4.00, PZ0.03, effect size f 2 Z0.17; TNF-a: F (1.71/66.81)Z4.75; PZ0.02; effect size f2Z 0.20). CFS patients showed an inverse response pattern in comparison to healthy controls, i.e. stimulated cytokine production decreased shortly after stress in CFS patients, while it increased in controls (Fig. 2). As glucocorticoids are known to
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Figure 1 ACTH, plasma cortisol and salivary cortisol responses over time and integrated endocrine responses (inserted bar graphs) of CFS patients (& or black) and healthy controls (C or gray) in the TSST.
exert an inhibitory influence on the secretion of pro-inflammatory cytokines, AUC of plasma and salivary free cortisol responses in the TSST were included as covariates in a subsequent ANCOVA for repeated measures. No differences in the level of significance and effect size measures were observed (PC: Il-6: F (1.63/62.82)Z3.93, PZ0.03,
effect size f2Z0.16; TNF-a: F (1.73/65.19)Z4.64; PZ0.02; effect size f2Z0.20; SC: Il-6: F (1.67/ 63.65)Z4.66, PZ0.02, effect size f2Z0.19; TNF-a: F (1.71/64.91)Z4.80; PZ0.02; effect size f2Z 0.20), indicating that absolute individual salivary free cortisol responses were not related to the differences in pro-inflammatory cytokine responses.
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Table 2
Immune responses in the TSST: changes of leukocyte and monocyte numbers after stress.
Parameter
Group
Samplea Basal
10 min after TSST
60 min after TSST
Leukocytes (!109 cells/l)
CFS Controls
5.98 (0.40) 5.85 (0.24)
6.10 (0.39) 5.91 (0.23)
6.17 (0.33) 6.16 (0.25)
Monocytes (!106 cells/l)
CFS Controls
451.82 (18.89) 478.20 (23.20)
480.36 (19.24) 456.83 (29.04)
444.40 (23.76) 491.38 (29.11)
a
Mean (SEM).
3.4. Associations between immune parameters and fatigue level To assess possible associations between stimulated pro-inflammatory cytokines levels and fatigue symptoms, Pearson’s correlations between the baseline (pre-TSST) as well as AUC of both cytokine responses curves and the scales of the FS were calculated for both groups separately (Table 3). For the control group, baseline levels of LPS-induced Il-6, but not TNF-a, were correlated with fatigue symptoms. The integrated cytokine responses in the TSST were not associated with fatigue levels in controls. In CFS patients, both baseline cytokine levels were significantly associated with fatigue scores. In addition, integrated cytokine responses in the TSST were significantly correlated with fatigue levels in the patients group. Correlation coefficients differed significantly between groups for baseline TNF-alpha and all integrated cytokine responses, but not for baseline Il-6 (data not shown). To examine whether significant correlations between AUC of cytokine responses and fatigue levels in CFS patients were a consequence of the existing association between baseline cytokine and fatigue levels, partial correlations between AUC of cytokine responses and fatigue levels controlling for baseline cytokine levels were performed. None of the correlations between AUC of cytokine responses and fatigue levels in CFS patients remained significant (data not shown).
response differences in cortisol parameters (Gaab et al., 2002a). In comparison to healthy controls, CFS patients were characterized by an inverted stimulated pro-inflammatory cytokine response during stress. Patients had comparable levels of LPS-induced Il-6 and TNF-a before and 60 min after the stress test, but unlike healthy controls, who showed a stress-induced increase of stimulated pro-inflammatory cytokine levels, CFS patients showed a decline in these parameters. The finding
4. Discussion This study set out to assess stimulated proinflammatory cytokine responses during psychosocial stress in CFS patients and healthy controls matched for age and gender. As previously reported, CFS patients had a significantly reduced ACTH response in the psychosocial stress test, which was not followed by similar
Figure 2 Pro-inflammatory cytokine responses of CFS patients (&) and healthy controls (C) in the TSST.
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Table 3 Pearson’s correlation between fatigue Scale total score and subscales and AUC of the stimulated proinflammatory cytokine responses in the TSST (significant correlations in bold). Baseline Il-6a
Baseline TNF-aa
AUC of Il-6a
AUC of TNF-aa
FS total score
CFS patients Control group
0.42, PZ0.03 0.45, PZ0.02
0.69, PZ0.000 0.12, PZ0.31
0.46, PZ0.02 0.18, PZ0.22
0.60, PZ0.002 0.16, PZ0.25
FS mental fatigue
CFS patients Control group
0.40, PZ0.04 0.49, PZ0.01
0.51, PZ0.001 0.15, PZ0.26
0.26, PZ0.13 0.16, PZ0.25
0.40, PZ0.04 0.16, PZ0.25
FS physical fatigue
CFS patients Control group
0.36, PZ0.05 0.39, PZ0.05
0.63, PZ0.001 0.08, PZ0.37
0.51, PZ0.01 0.19, PZ0.21
0.58, PZ0.003 0.16, PZ0.25
a
LPS-induced cytokine levels.
of short-term stress-induced increases in LPSstimulated pro-inflammatory cytokine levels in healthy subjects replicates the results of previous studies reporting increases of LPS-induced Il-6 and TNF-a after psychological or physical stress (Maes et al., 1998; Goebel et al., 2000). Groups did not differ with regard to total number of leukocytes and monocytes during and after the stress test. Furthermore, as indicated by the results of the ANCOVA, this inverse response pattern was not caused by possible differences in the individual absolute stress-induced cortisol responses. Calculating the associations between immune parameters and fatigue scores for each group separately, high correlations were found for baseline levels of Il-6 in both groups, whereas for baseline TNF-a, high correlations were found in CFS patients, but not in controls. Accordingly, only in CFS patients were integrated cytokine responses significantly correlated with fatigue symptoms. However, partial correlations revealed that these effects were a consequence of the high correlations between fatigue and cytokine levels at baseline. The results indicate that in the population under study, although cytokine responses were normal or attenuated, overall stimulated pro-inflammatory cytokine release is associated with general fatigue levels in CFS. However, it should be noted that this effect does not seem to be mediated through an exaggerated stress-induced increase in stimulated cytokine secretion. The observed cytokine response pattern does not appear to be caused by a possibly enhanced inhibition of cytokine production due to an exaggerated secretion of either plasma or salivary cortisol responses, since cortisol responses do not differ significantly between CFS patients and controls. With regard to inhibitory mechanisms of LPSinduced pro-inflammatory cytokine production, two important processes can be identified. On the one hand, glucocorticoids are known to inhibit the release of pro-inflammatory cytokines, thus promoting a shift from Th 1- to Th 2-dependent or cellular to humoral immune response, while
the effects of catecholamines are more heterogeneous. In-vitro, catecholamines suppress proinflammatory cytokines (Elenkov et al., 1996), while in-vivo, catecholamines may also activate cytokine production, for example via induction of NF-kB (Bierhaus, 2003, see below). It could be reasoned that the observed cytokine response differences are possibly not due to quantitative differences of the respective parameter, but rather to qualitative differences. From this point of view, it would not be the absolute amount that is important, but rather its relative effect. In CFS, a number of studies using different methods and investigating different target cells provide evidence for a general enhanced sensitivity to glucocorticoids, e.g. cytokine-specific supersensitivity of purified CD4 T cells to dexamethasone (Visser et al., 2001b), an enhanced sensitivity of pituitary corticotrophs to the inhibiting effects of 0.5 mg dexamethasone (Gaab et al., 2002b), an enhanced adrenal cortisol output to low but not to high levels of ACTH (Demitrack et al., 1991; Gaab et al., 2002a; Gaab et al., 2003b), and significant reduction of fatigue symptoms with treatment of 5–10 mg, but not 25–35 mg hydrocortisone (McKenzie et al., 1998; Cleare et al., 1999). In contrast, Kavelaars et al. (2000) observed a diminished capacity of dexamethasone to inhibit PHA-induced T cell proliferation, although the inhibitory concentration needed to achieve a 50% for DEX inhibition of PHA-induced cytokine production (IC50) was normal in CFS. This latter finding is in contrast to findings of enhanced glucocorticoid sensitivity for DEX inhibition of LPS-induced cytokine production, indicated by lower doses of DEX required for 50% inhibition of the maximum cytokine production observed after LPS stimulation without DEX (Gaab et al., 2003c), and an enhanced sensitivity of PHA-stimulated CD4 T cell and purified peripheral blood mononuclear cell proliferation to the suppressive effects of glucocorticoids (Visser et al.,1998, 2001b). These differences
196 could partly be explained by differences in the methods and subjects used, since Kavelaars et al. employed their methods in adolescent girls with CFS, while the contrasting studies were performed on adult patient populations. With regard to alteration of the sensitivity towards catecholamines, a reduced capacity of the b2-adrenergic agonist terbutaline to regulate the production of TNF-alpha and IL-10 has been reported in adolescent female CFS patients (Kavelaars et al., 2000). The exact mechanisms of the findings observed in our study and in the studies reported above remain to be elucidated, but given that the affinity, the number of GR, and the GR messenger RNA expression have been shown to be normal in CFS (Visser et al., 2001b), qualitative dysregulations of intracellular mechanism, e.g. the transduction of the signal from the receptor to the intracellular effector system, could be a possible candidate. The transcription factor nuclear factor (NF)-kB is a crucial signal for activation of the cellular inflammatory mechanism, e.g. cytokine production, adhesion molecule expression, etc. (McKay and Cidlowski, 1999). NF-kB is activated not only by cellular stress and inflammation signals, but also by psychosocial stress to which the whole organism is exposed, mediated via alpha-2 and beta-adrenergic receptors (Bierhaus et al., 2003). Glucocorticoids exert their inhibitory effects on cytokine production via suppression of NF-kB DNAbinding activity. Changes in the sensitivity of cells towards glucocorticoid inhibition can be mediated via a large array of extra- and intracellular mechanisms (Rohleder et al., 2003). In CFS patients, a decreased NF-kB activity in response to stress could be a possible intracellular mechanism to mediate the assumed increased glucocorticoid sensitivity. This would also be compatible with a decrease of cytokine production after stress. Attenuated catecholamine response to stress (which is unlikely, as this appears to be normal in CFS (Gaab et al., 2003a)), or lower sensitivity of cells towards catecholamines, for example due to a disturbed adrenoceptor balance (Kavelaars et al., 2000), could contribute to a decreased NF-kB activation by stress. However, these assumptions need to be tested in further studies. In addition, other mechanisms are also possible. With regard to the difference between patients and controls in the correlational pattern between LPS-induced pro-inflammatory cytokine secretion and fatigue levels, it needs to be noted that Il-6 has been shown to induce fatigue symptoms in healthy subjects and CFS patients (Arnold et al., 2002).
J. Gaab et al. However, in contrast to controls, CFS patients’ general fatigue levels were also strongly correlated with baseline LPS-induced TNF-a levels. From a somatic perspective, it has been suggested that cytokines, besides immediate effects, have protracted consequences through the promotion of behavioral and neurochemical sensitization (Anisman and Merali, 2003). Thus, behavioral responses to subsequent cytokine challenges are enhanced. For example, systemic TNF-a administration proactively enhanced the induction of sickness behavior to subsequent administration of the cytokine in mice (Hayley et al., 1999). In a recent study employing an Il-6 challenge to induce influenza-like symptoms, CFS patients experienced a greater amount of somatic symptoms with a shorter latency in comparison to healthy controls (Arnold et al., 2002). In CFS, stress, due either to psychiatric disorders, critical life events, or symptom-related distress, could lead to repeated secretion of pro-inflammatory cytokines, resulting in the previously described sensitization process. It has been suggested that “further increases in distress as a ‘reaction’ to mounting symptoms creates a vicious cycle” and that ’such a recursive system may act as a positive feedback loop, accounting for the chronic nature of CFS‘ (cited from: (Patarca, 2001), page 194). In conclusion, CFS patients did not show exaggerated pro-inflammatory cytokine responses during stress, but rather the opposite was the case. An enhanced sensitivity to the suppressive effects of glucocorticoids could serve as a possible physiological explanation for this finding. LPSinduced cytokine levels were strongly correlated with general fatigue levels in CFS patients, although cytokine levels were comparable to those of controls. An enhanced perception of cytokineinduced symptoms, possibly due to sensitization processes, could serve as an explanatory model to account for these findings. Further studies are needed to examine the role of stress-induced pro-inflammatory cytokine release in the perpetuation of the syndrome. For example, it would be interesting to evaluate the effects of interventions that aim to reduce stress and change symptom perception and attribution, such as cognitive behavioral therapy, on the correlation between pro-inflammatory cytokine secretion and CFS symptoms. In addition, it would be worth expanding the focus not only on pro-inflammatory cycles, but also on anti-inflammatory cytokines, since it has been shown that glucocorticoid regulation of pro- and anti-inflammatory cytokines is altered in CFS patients (Visser et al., 2000).
Inflammatory cytokines and CFS
References Anisman, H., Merali, Z., 2003. Cytokines, stress and depressive illness: brain-immune interactions. Ann. Med. 35, 2–11. Arnold, M.C., Papanicolaou, D.A., O’Grady, J.A., Lotsikas, A., Dale, J.K., Straus, S.E., Grafman, J., 2002. Using an interleukin-6 challenge to evaluate neuropsychological performance in chronic fatigue syndrome. Psychol. Med. 32, 1075–1089. Bierhaus, A., Wolf, J., Andrassy, M., Rohleder, N., Humpert, P.M., Petrov, D., Ferstl, R., von Eynatten, M., Wendt, T., Rudofsky, G., Joswig, M., Morcos, M., Schwaninger, M., McEwen, B., Kirschbaum, C., Nawroth, P.P., 20031. A mechanism converting psychosocial stress into mononuclear cell activation. Proc. Natl. Acad. Sci. USA 100, 1920–1925. Cannon, J.G., Angel, J.B., Abad, L.W., O’Grady, J., Lundgren, N., Fagioli, L., Komaroff, A.L., 1998. Hormonal influences on stress-induced neutrophil mobilization in health and chronic fatigue syndrome. J. Clin. Immunol. 18, 291–298. Chalder, T., Berelowitz, G., Pawlikowska, T., Watts, L., Wessely, S., Wright, D., Wallace, E.P., 1993. Development of a fatigue scale. J. Psychosom. Res. 37, 147–153. Clauw, D.J., Chrousos, G.P., 1997. Chronic pain and fatigue syndromes: overlapping clinical and neuroendocrine features and potential pathogenic mechanisms. Neuroimmunomodulation 4, 134–153. Cleare, A.J., 2003. The neuroendocrinology of chronic fatigue syndrome. Endocr. Rev. 24, 236–252. Cleare, A.J., Heap, E., Malhi, G.S., Wessely, S., O’Keane, V., Miell, J., 1999. Low-dose hydrocortisone in chronic fatigue syndrome: a randomised crossover trial. Lancet 353, 455–458. Cleeland, C.S., Bennett, G.J., Dantzer, R., Dougherty, P.M., Dunn, A.J., Meyers, C.A., Miller, A.H., Payne, R., Reuben, J.M., Wang, X.S., Lee, B.N., 2003. Are the symptoms of cancer and cancer treatment due to a shared biologic mechanism? A cytokine-immunologic model of cancer symptoms. Cancer 97, 2919–2925. Dantzer, R., 2001. Cytokine-induced sickness behavior: where do we stand?. Brain Behav. Immun. 15, 7–24. Demitrack, M.A., Dale, J.K., Straus, S.E., Laue, L., Listwak, S.J., Kruesi, M.J., Chrousos, G.P., Gold, P.W., 1991. Evidence for impaired activation of the hypothalamic-pituitary-adrenal axis in patients with chronic fatigue syndrome. J. Clin. Endocrinol. Metab. 73, 1224–1234. Dressendorfer, R.A., Kirschbaum, C., Rohde, W., Stahl, F., Strasburger, C.J., 1992. Synthesis of a cortisol-biotin conjugate and evaluation as a tracer in an immunoassay for salivary cortisol measurement. J. Steroid. Biochem. Mol. Biol. 43, 683–692. Elenkov, I.J., Papanicolaou, D.A., Wilder, R.L., Chrousos, G.P., 1996. Modulatory effects of glucocorticoids and catecholamines on human interleukin-12 and interleukin-10 production: clinical implications. Proc. Assoc. Am. Physicians 108, 374–381. Franke, G., 1995. Die Symptom-Checkliste von DerogatisDeutsche Version-Manual (2., rev. u. erw. Aufl.). Beltz Test GmbH, Weinheim. Fukuda, K., Straus, S.E., Hickie, I., Sharpe, M.C., Dobbins, J.G., Komaroff, A., 1994. The chronic fatigue syndrome: a comprehensive approach to its definition and study. International Chronic Fatigue Syndrome Study Group. Ann. Intern. Med. 121, 953–959.
197 Gaab, J., Huster, D., Peisen, R., Engert, V., Heitz, V., Schad, T., Schurmeyer, T.H., Ehlert, U., 2002a. Hypothalamic-pituitaryadrenal axis reactivity in chronic fatigue syndrome and health under psychological, physiological, and pharmacological stimulation. Psychosom. Med. 64, 951–962. Gaab, J., Huster, D., Peisen, R., Engert, V., Schad, T., Schurmeyer, T.H., Ehlert, U., 2002b. Low-dose dexamethasone suppression test in chronic fatigue syndrome and health. Psychosom. Med. 64, 311–318. Gaab, J., Engert, V., Schad, T., Heitz, V., Schu ¨rmeyer, T., Ehlert, U., 2003a. Catecholaminergic responsiveness under physiological stress in chronic fatigue syndrome. Psychosom. Med. 61, A26. Gaab, J., Huster, D., Peisen, R., Engert, V., Heitz, V., Schad, T., Schurmeyer, T., Ehlert, U., 2003b. Assessment of cortisol response with low-dose and high-dose ACTH in patients with chronic fatigue syndrome and healthy comparison subjects. Psychosomatics 44, 113–119. Gaab, J., Rohleder, N., Heitz, V., Engert, V., Schad, T., Schu ¨rmeyer, T., Ehlert, U., 2003c. Enhanced glucocorticoid sensitivity in patients with chronic fatigue syndrome. Acta Neuropsychiatr. 15, 184–191. Goebel, M.U., Mills, P.J., Irwin, M.R., Ziegler, M.G., 2000. Interleukin-6 and tumor necrosis factor-alpha production after acute psychological stress, exercise, and infused isoproterenol: differential effects and pathways. Psychosom. Med. 62, 591–598. Hayley, S., Brebner, K., Lacosta, S., Merali, Z., Anisman, H., 1999. Sensitization to the effects of tumor necrosis factoralpha: neuroendocrine, central monoamine, and behavioral variations. J. Neurosci. 19, 5654–5665. Herrmann, C., Buss, U., Snaith, R.P., 1995. HADS-D Hospital Anxiety and Depression Scale-Ein Fragebogen zur Erfassung von Angst und Depressivita ¨t in der somatischen Medizin. Hans Huber 1995. Kavelaars, A., Kuis, W., Knook, L., Sinnema, G., Heijnen, C.J., 2000. Disturbed neuroendocrine-immune interactions in chronic fatigue syndrome. J. Clin. Endocrinol. Metab. 85, 692–696. Kessler, S., Jaeckel, W., Cziske, R., 1997. Assessing health in musculoskeletal disorders–the appropriateness of a German version of the sickness impact profile. Rheumatol. Int. 17, 119–125. Kirschbaum, C., Pirke, K.M., Hellhammer, D.H., 1993. The ‘Trier Social Stress Test’—a tool for investigating psychobiological stress responses in a laboratory setting. Neuropsychobiology 28, 76–81. LaManca, J.J., Sisto, S.A., Zhou, X.D., Ottenweller, J.E., Cook, S., Peckerman, A., Zhang, Q., Denny, T.N., Gause, W.C., Natelson, B.H., 1999. Immunological response in chronic fatigue syndrome following a graded exercise test to exhaustion. J. Clin. Immunol. 19, 135–142. Lariviere, W.R., Melzack, R., 2000. The role of corticotropinreleasing factor in pain and analgesia. Pain 84, 1–12. Leese, G., Chattington, P., Fraser, W., Vora, J., Edwards, R., Williams, G., 1996. Short-term night-shift working mimics the pituitary-adrenocortical dysfunction in chronic fatigue syndrome. J. Clin. Endocrinol. Metab. 81, 1867– 1870. Lloyd, A., Gandevia, S., Brockman, A., Hales, J., Wakefield, D., 1994. Cytokine production and fatigue in patients with chronic fatigue syndrome and healthy control subjects in response to exercise. Clin. Infect. Dis. 18, S142–S146. Luger, A., Deuster, P.A., Kyle, S.B., Gallucci, W.T., Montgomery, L.C., Gold, P.W., Loriaux, D.L., Chrousos, G.P.,
198 1987. Acute hypothalamic-pituitary-adrenal responses to the stress of treadmill exercise. Physiologic adaptations to physical training. N. Engl. J. Med. 316, 1309–1315. Lutgendorf, S.K., Antoni, M.H., Ironson, G., Fletcher, M.A., Penedo, F., Baum, A., Schneiderman, N., Klimas, N., 1995. Physical symptoms of chronic fatigue syndrome are exacerbated by the stress of Hurricane Andrew. Psychosom. Med. 57, 310–323. Maes, M., Song, C., Lin, A., De Jongh, R., Van Gastel, A., Kenis, G., Bosmans, E., De Meester, I., Benoy, I., Neels, H., Demedts, P., Janca, A., Scharpe, S., Smith, R.S., 1998. The effects of psychological stress on humans: increased production of proinflammatory cytokines and a Th1-like response in stressinduced anxiety. Cytokine 10, 313–318. McEwen, B.S., Biron, C.A., Brunson, K.W., Bulloch, K., Chambers, W.H., Dhabhar, F.S., Goldfarb, R.H., Kitson, R.P., Miller, A.H., Spencer, R.L., Weiss, J.M., 1997. The role of adrenocorticoids as modulators of immune function in health and disease: neural, endocrine and immune interactions. Brain Res. Brain Res. Rev. 23, 79–133. McKay, L.I., Cidlowski, J.A., 1999. Molecular control of immune/inflammatory responses: interactions between nuclear factor-kappa B and steroid receptor-signaling pathways. Endocr. Rev. 20, 435–459. McKenzie, R., O’Fallon, A., Dale, J., Demitrack, M., Sharma, G., Deloria, M., Garcia-Borreguero, D., Blackwelder, W., Straus, S.E., 1998. Low-dose hydrocortisone for treatment of chronic fatigue syndrome: a randomized controlled trial. J. Am. Med. Assoc. 280, 1061–1066. Natelson, B.H., 2001. Chronic fatigue syndrome. J. Am. Med. Assoc. 285, 2557–2559. Papanicolaou, D.A., Petrides, J.S., Tsigos, C., Bina, S., Kalogeras, K.T., Wilder, R., Gold, P.W., Deuster, P.A., Chrousos, G.P., 1996a. Exercise stimulates interleukin-6 secretion: inhibition by glucocorticoids and correlation with catecholamines. Am. J. Physiol. 271, E601–E605. Papanicolaou, D.A., Tsigos, C., Oldfield, E.H., Chrousos, G.P., 1996b. Acute glucocorticoid deficiency is associated with plasma elevations of interleukin-6: does the latter participate in the symptomatology of the steroid withdrawal syndrome and adrenal insufficiency?. J. Clin. Endocrinol. Metab. 81, 2303–2306. Patarca, R., 2001. Cytokines and chronic fatigue syndrome. Ann. N Y Acad. Sci. 933, 185–200. Peterson, P.K., Sirr, S.A., Grammith, F.C., Schenck, C.H., Pheley, A.M., Hu, S., Chao, C.C., 1994. Effects of mild exercise on cytokines and cerebral blood flow in chronic fatigue syndrome patients. Clin. Diagn. Lab. Immunol. 1, 222–226. Pruessner, J.C., Kirschbaum, C., Meinlschmid, G., Hellhammer, D.H., 2003. Two formulas for computation of the area under the curve represent measures of total hormone concentration versus time-dependent change. Psychoneuroendocrinology 28, 916–931.
J. Gaab et al. Rohleder, N., Wolf, J.M., Kirschbaum, C., 2003. Glucocorticoid sensitivity in humans-interindividual differences and acute stress effects. Stress 6, 207–222. Sharpe, M.C., Archard, L.C., Banatvala, J.E., Borysiewicz, L.K., Clare, A.W., David, A., Edwards, R.H., Hawton, K.E., Lambert, H.P., Lane, R.J., et al., 1991. A report—chronic fatigue syndrome: guidelines for research. J.R. Soc. Med. 84, 118–121. Steptoe, A., Willemsen, G., Owen, N., Flower, L., MohamedAli, V., 2001. Acute mental stress elicits delayed increases in circulating inflammatory cytokine levels. Clin. Sci. (Lond) 101, 185–192. Theorell, T., Blomkvist, V., Lindh, G., Evengard, B., 1999. Critical life events, infections, and symptoms during the year preceding chronic fatigue syndrome (CFS): an examination of CFS patients and subjects with a nonspecific life crisis. Psychosom. Med. 61, 304–310. Vgontzas, A.N., Zoumakis, M., Papanicolaou, D.A., Bixler, E.O., Prolo, P., Lin, H.M., Vela-Bueno, A., Kales, A., Chrousos, G.P., 2002. Chronic insomnia is associated with a shift of interleukin-6 and tumor necrosis factor secretion from nighttime to daytime. Metabolism 51, 887–892. Visser, J., Blauw, B., Hinloopen, B., Brommer, E., de Kloet, E.R., Kluft, C., Nagelkerken, L., 1998. CD4 T lymphocytes from patients with chronic fatigue syndrome have decreased interferon-gamma production and increased sensitivity to dexamethasone. J. Infect. Dis. 177, 451–454. Visser, J., Graffelman, W., Blauw, B., Haspels, I., Lentjes, E., de Kloet, E.R., Nagelkerken, L., 2001a. LPS-induced IL-10 production in whole blood cultures from chronic fatigue syndrome patients is increased but supersensitive to inhibition by dexamethasone. J. Neuroimmunol. 119, 343–349. Visser, J., Lentjes, E., Haspels, I., Graffelman, W., Blauw, B., de Kloet, R., Nagelkerken, L., 2001b. Increased sensitivity to glucocorticoids in peripheral blood mononuclear cells of chronic fatigue syndrome patients, without evidence for altered density or affinity of glucocorticoid receptors. J. Investig. Med. 49, 195–204. Visser, J.T., De Kloet, E.R., Nagelkerken, L., 2000. Altered glucocorticoid regulation of the immune response in the chronic fatigue syndrome. Ann. NY Acad. Sci. 917, 868–875. Vollmer-Conna, U., Lloyd, A., Hickie, I., Wakefield, D., 1998. Chronic fatigue syndrome: an immunological perspective. Aust. NZJ Psychiatr. 32, 523–527. Wittchen, H.-U., Pfister, H., 1997. DIA-X Interviews: Manual fu ¨r Screening-Verfahren und Interview. Swets and Zeitlinger, Frankfurt. Wood, G.C., Bentall, R.P., Gopfert, M., Dewey, M.E., Edwards, R.H., 1994. The differential response of chronic fatigue, neurotic and muscular dystrophy patients to experimental psychological stress. Psychol. Med. 24, 357–364.
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Stress-induced changes in LPS-induced pro-inflammatory cytokine production in chronic fatigue syndrome Jens Gaab*, Nicolas Rohleder1, Vera Heitz, Veronika Engert, ¨rmeyer, Ulrike Ehlert2 Tanja Schad2, Thomas H. Schu Center for Psychobiological and Psychosomatic Research, University of Trier, Trier, Germany Received 18 March 2004; received in revised form 11 June 2004; accepted 12 June 2004
KEYWORDS Chronic fatigue syndrome; HPA axis; Psychosocial stress test; Pro-inflammatory cytokines; Il-6; TNF-a; ACTH; Cortisol; LPS
Summary Objective. It has been suggested that a hypofunctional hypothalamic– pituitary–adrenal (HPA) axis in chronic fatigue syndrome could result in an exaggerated release of pro-inflammatory cytokines during stress. As pro-inflammatory cytokines are involved in the induction of sickness behavior and thus constitute a potential physiological correlate of stress-induced symptom exacerbation in chronic fatigue syndrome, we set out to evaluate the LPS-induced production of proinflammatory cytokines during psychosocial stress in CFS and healthy controls. Method. Twenty-one CFS patients and 20 healthy controls matched for age and gender underwent a standardized psychosocial stress test (Trier social stress test, TSST). Adrenocorticotropine hormone (ACTH), salivary cortisol and plasma cortisol levels were measured before and repeatedly following exposure to the stressor. Lipopolysaccharide-stimulated production of interleukin-6 and tumor necrosis factor-alpha were assessed at baseline as well as 10 and 60 min after the stress test. Results. CFS patients showed an inverse stress-induced response pattern of LPSstimulated cytokines responses in comparison to healthy controls, i.e. stimulated cytokine production decreased shortly after stress in CFS patients, while it increased in controls. Fatigue scores and basal LPS-induced cytokine levels were significantly associated for TNF-a in controls and for both cytokines in CFS patients. Stressinduced changes in stimulated cytokine production were not associated with general fatigue scores in the control group, whereas in the CFS group, fatigue scores were significantly correlated with integrated levels of LPS-induced cytokines.
Abbreviations: CFS, chronic fatigue syndrome; ACTH, adrenocorticotropine hormone; PC, plasma cortisol; SC, saliva cortisol; HR, heart rate; TSST, Trier social stress test; CLIA, chemiluminscence assay; SIP, sickness impact profile; HADS, hospital anxiety and depression scale; SCL-90R, symptom checklist; AUC, area under curve. * Corresponding author. Present address: Institute for Psychology, Clinical Psychology and Psychotherapy, University of Zu ¨rich, Zu ¨richbergstr. 43, Zu ¨rich, Switzerland. Tel.: C41-1-6343096; fax: C41-1-6343696. E-mail address: [email protected] (J. Gaab). 1 Present address: Department of Biopsychology, Technical University of Dresden, Dresden, Germany. 2 Present address: Institute for Psychology, Clinical Psychology and Psychotherapy, University of Zu ¨rich, Zu ¨richbergstr. 43, Zu ¨rich, Switzerland. 0306-4530/$ - see front matter Q 2004 Elsevier Ltd. All rights reserved.
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However, partial correlations revealed that these results were due to the high correlations with basal LPS-induced cytokine levels. Conclusion. CFS patients do not show an exaggerated secretion of LPS-induced cytokines. Although cortisol responses to stress were normal, pro-inflammatory cytokine levels in CFS patients were significantly attenuated. Possible intracellular mechanisms, such as for example an enhanced sensitivity to inhibitory effects of glucocorticoids, a diminished responsivity to catecholaminergic stimulation, and a disruption of intracellular activation are discussed. Basal levels of stimulated proinflammatory Il-6 levels are generally related to fatigue scores. However, in CFS patients this association is of greater magnitude and can also be observed for TNF-a. Q 2004 Elsevier Ltd. All rights reserved.
1. Introduction Chronic fatigue syndrome (CFS) is characterized by persistent or relapsing debilitating mental and physical fatigue, which is exacerbated by minor exertion. Patients also experience further somatic symptoms, such as myalgia, arthralgia, cognitive disturbances, low-grade fever, and sleep disturbances (Fukuda et al., 1994). Although these symptoms are indicative of a somatic origin, and despite a large number of empirical studies, no objective and specific somatic pathology has been identified. Consequently, the diagnosis of CFS is established by the exclusion of somatic and psychiatric causes of chronic fatigue. However, dysregulations of somatic systems involved in the maintenance of homeostasis have been observed and proposed as physiological substrates of symptoms experienced (Natelson, 2001). Amongst other candidates, the interaction of psychological, endocrine, and immune processes seems to be of particular relevance, since on the one hand psychological and behavioral factors associated with the precipitation and perpetuation of the syndrome, such as stress, inactivity and sleep problems (Luger et al., 1987; Leese et al., 1996; Theorell et al., 1999), are also known to influence endocrine and consequently immune functioning (for review: (McEwen et al., 1997) and on the other hand, alterations of neuroendocrine and immune mechanisms can be linked to CFS symptoms (for review: (Clauw and Chrousos, 1997; Lariviere and Melzack, 2000; Dantzer, 2001). From this perspective, alterations of the bidirectional communication between the hypothalamic–pituitary–adrenal axis and the immune system or, more precisely, between glucocorticoids and cytokines have been discussed as a possible etiological model for CFS (for review: (VollmerConna et al., 1998; Patarca, 2001). In general, glucocorticoids modulate the immune response by inhibiting the production and release of
pro-inflammatory cytokines, thus promoting the switch from a T helper (Th) 1- to Th 2-dependent or cellular to humoral immune response respectively. With regard to CFS, it is noteworthy that proinflammatory cytokines are mediators of the behavioral and motivational changes seen during infections, referred to as ’sickness behavior’ (Dantzer, 2001), and elevated levels of or treatment with pro-inflammatory cytokines have been shown to be associated with fatigue symptomatology (Papanicolaou et al., 1996b; Vgontzas et al., 2002; Cleeland et al., 2003). Since pro-inflammatory cytokines are released during psychosocial and physiological stress (Papanicolaou et al., 1996a; Goebel et al., 2000; Steptoe et al., 2001), a hypersecretion of pro-inflammatory cytokines during stress due to a hypofunctional neuroendocrine counterregulation could serve as a possible explanation of exercise- and stress-induced exacerbation of fatigue experienced by patients with CFS (Wood et al., 1994; Lutgendorf et al., 1995). Several studies have investigated dysregulations of the HPA axis (for review: (Cleare, 2003) and alterations of circulating cytokine levels or stimulated cytokine production in CFS (for review: (Visser et al., 2000; Patarca, 2001). However, findings of HPA axis dysregulations and alterations of cytokine levels and production are heterogeneous, possibly reflecting both the heterogeneity of the subjects under study with regard to duration of symptoms, inactivity, sleep disturbance, psychiatric comorbidity, medication, and ongoing stress as well as the heterogeneity of methods and study designs used. With regard to the interaction of neuroendocrine parameters and pro-inflammatory cytokine production in CFS, Visser and colleagues reported supersensitivity of lipopolysaccharide (LPS) -induced IL-10 and IFN-g, but not of IL-12 or IL-4 production to inhibition by dexamethasone, independent of the number or affinity of glucocorticoid receptors (GR) (Visser et al., 1998, 2001a,b).
190 A number of studies have assessed immune responses during or after stress in CFS. Cannon et al. (Cannon et al., 1998) reported a disrupted endocrine influence on neutrophil mobilization in CFS patients undergoing a standardized exercise program, without indication of an excessive immune response. Other studies employing mild exercise tests were unable to find significant differences in cytokine levels between CFS patients and controls (Lloyd et al., 1994; Peterson et al., 1994; LaManca et al., 1999). In contrast to these studies using physical exercise tests, recent reports suggest an enhanced sensitivity of circulating leucocytes to increasing doses of dexamethasone in CFS patients undergoing a standardized psychosocial stress paradigm (Gaab et al., 2003c). In summary, the results of previous studies on this topic are either conflicting or do not support the assumption on an attenuated suppression of pro-inflammatory cytokine responses by glucocorticoids. However, the hypothesis that an altered neuroendocrine-immune interplay during stress could be used to explain fatigue symptoms has not been tested directly as yet. We therefore set out to assess the LPS-induced production of pro-inflammatory cytokines before and after a standardized psychosocial stress test in CFS patients and healthy controls and related these findings to HPA axis responses and general fatigue symptoms.
J. Gaab et al. and no medical cause for the chronic fatigue in routine laboratory testing. All patients were medically examined by the same physician (THS), according to recommendation (Fukuda et al., 1994), and were also interviewed by a trained psychologist (JG). This consisted of a computeraided standardized and structured diagnostic interview in accordance with the Diagnostic and Statistical Manual of Mental Health Disorders 4th Edition (Wittchen and Pfister, 1997) and a semistructured CFS interview, which was concerned with the severity and course of all symptoms required by the US and UK definitions (Sharpe et al., 1991; Fukuda et al., 1994). All patients fulfilled US and UK consensus criteria for the diagnosis of CFS (Sharpe et al., 1991; Fukuda et al., 1994). Patients were matched for age and gender with healthy volunteer controls, randomly recruited via telephone calls. Controls were free of medication and underwent comprehensive medical examination for past and current health problems. Control subjects were screened for any current or lifetime psychiatric symptoms or disorders by a clinical psychologist (JG). After subjects were provided with complete written and oral descriptions of the study, written informed consent was obtained. The study sample is part of a larger CFS patient cohort, the results of which have been published elsewhere (Gaab et al., 2002a,b).
2.2. Procedure
2. Methods 2.1. Subjects The study was approved by the Ethics Committee of the Medical Council of Rheinland-Pfalz, Germany. Patients were contacted through a German selfhelp organization. Interested parties received a postal screening questionnaire, containing all symptoms required by the UK and US definitions of CFS (Sharpe et al., 1991; Fukuda et al., 1994). Patients fulfilling the symptom requirements in the screening questionnaire were interviewed over the telephone and asked to disclose any diagnosed medical illnesses and psychiatric disorders. Prospective participants were only excluded from the study if they had received a medical or psychiatric diagnosis defined as an exclusion criterion by the US definition (Fukuda et al., 1994). Selection criteria for participation in the study were fulfillment of CFS symptom criteria in the postal screening questionnaire, new or definite onset of CFS, age between 30–50, no current antidepressant, anxiolytic, antibiotic, antihypertensive, and steroid medication
2.2.1. Trier social stress test (TSST) The TSST has been repeatedly found to induce profound endocrine and cardiovascular responses in 70–80% of the subjects tested (Kirschbaum et al., 1993). After basal blood and saliva samples were taken, subjects were introduced to the TSST (1 min). They were given 3 min to prepare themselves for a fake job interview (5 min), followed by a mental arithmetic task in front of an audience (5 min). Subjects were told that they would be videotaped for further analysis of their behavior. Immediately after the test, a blood and saliva sample was taken, with further blood and saliva samples taken at 10, 20, 30, 45, and 60 min to assess endocrine parameters. Blood was drawn into 9 ml ethylenediamine tetraacetate (EDTA) monovettes (Sarstedt, Nu ¨mbrecht, Germany). Additional samples were taken immediately before as well as 10 and 60 min after TSST for stimulation of inflammatory cytokine production into heparinized sterile syringes (Braun, Melsungen, Germany) and for a differential blood count into 2.3 ml EDTA monovettes (Sarstedt, Nu ¨mbrecht, Germany).
Inflammatory cytokines and CFS
2.3. Measures 2.3.1. Sampling methods and biochemical analyses EDTA blood samples for endocrine measures were spun immediately at 4 8C and stored at K20 8C until assayed. Saliva was collected by the subjects themselves using Salivette collection devices (Sarstedt, Nu ¨mbrecht, Germany) and stored at room temperature until completion of the session. It was then stored at K20 8C until biochemical analysis was carried out. ACTH and PC were measured with two-site commercial chemiluminescence assays (CLIA, Nichols Institute Diagnostics, Bad Nauheim, Germany). The free cortisol concentration in saliva (SC) was determined using a time-resolved immunoassay with fluorometric detection, as described in detail elsewhere (Dressendorfer et al., 1992). Inter- and intraassay coefficients of variance were below 10% for all analytes. For the assessment of inflammatory cytokines, whole blood was coincubated with LPS (E. Coli, Difco, Augsburg, Germany) on a 24-well plate (Greiner, Nuertingen, Germany). Diluted whole blood (400 ml) was added to 50 ml of LPS. The final concentration was 30 ng/ml LPS. After 18 h of incubation at 37 8C and 5% CO2, the plates were centrifuged for 10 min at 2000 g at 4 8C. The plasma supernatant was collected and stored at K80 8C until assayed. IL-6 and TNF-alpha were measured using ELISA, employing the multiple antibody sandwich principle (BD Pharmingen, San Diego, CA, USA). Plates were read by a microplate reader (Anthos HTII, Anthos Labtec, Salzburg, Austria), and absorbance was transformed to cytokine concentration (pg/ml) using a standard curve computed by Anthos Winread 2.3 software (Anthos Labtec, Salzburg, Austria). Inter- and intraassay coefficients of variation were below 10%. To correct for changing numbers of monocytes, cytokine levels are expressed as ng relative to one million monocytes. A differential blood count was performed, measuring absolute numbers of leukocytes, as well as number and percentage of monocytes, lymphocytes, and granulocytes. Cell counting was performed on a Coulter AcTdiff cell counter (BeckmanCoulter, Krefeld, Germany). 2.3.2. Psychometric measures and patient characteristics To assess the severity of the most prominent CFS symptoms, all subjects completed a German translation of the fatigue scale (FS) (Chalder et al., 1993). To be able to assess the general fatigue
191 severity, the FS was administered at least one day before the psychosocial stress test. The FS is an 11-item self-report measure developed to assess fatigue. It consists of two scales, which assess physical and mental fatigue. We used a 0, 1, 2, 3 scoring system and calculated a total score. An internal consistency (Cronbach’s alpha) of aZ0.96 for the total score has been calculated on a larger CFS population (NZ193) (Gaab et al., unpublished data). In addition, all subjects completed a battery of questionnaires, including the sickness impact profile (SIP) (Kessler et al., 1997), the hospital anxiety and depression scale (HADS), which measures symptoms occurring during the last week (Herrmann et al., 1995), and the symptom checklist (SCL-90R), which assesses symptoms experienced during the last four weeks (Franke, 1995). All of these scales have been evaluated on German patient populations, with good validity and reliability. Duration of CFS was assessed in months since onset of symptoms.
2.4. Statistical analysis c2 analysis was used to test for significant differences in discrete variables. ANOVAS were computed to analyze endocrine and immune parameters between groups. Correlations were computed as Pearson product-moment correlations and partial correlations. For all endocrine parameters, areas under the total response curve (AUC), expressed as area under all samples, were calculated using the trapezoidal method (Pruessner et al., 2003). To test for possible differences between correlations, coefficients were Fisher-Z transformed and tested for significance using z-distribution tables. KolmogorovSmirnov tests revealed that immune data was not normally distributed. Calculating the log of cytokine values produced nearly normally distributed values. Although log-transformed cytokine values were used for all statistical analyses, means and standard deviations of untransformed values are presented. Data was also tested for homogeneity of variance using Levene’s test before statistical procedures were applied. For all analyses, significance levels were aZ5%. Unless indicated, all results shown are meansGstandard error of means (SEM).
3. Results 3.1. Sample characteristics Twenty-one CFS patients and 20 controls participated in the study. Gender ratio, number of
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Table 1
Demographic and psychometric characteristics of CFS patients and healthy controls.
Sex (male/female) TSST Age in yearsa BMIa Fatigue scale HADSb Depression Anxiety SCL-90Rb Anxiety Phobic anxiety Depression Somatization SIPb Home management Ambulation Mobility Alertness behavior Sleep and rest Social interaction a b
CFS
Controls
10/11 36.00 (30–47) 22.4 (17.6–26.8) 25.76 (0.95)
11/9 35.24 (29–44) 24.1 (18.1–34.6) 9.45 (0.98)
7.3 (0.9) 6.4 (0.9)
1.4 (0.3) 3.2 (0.5)
58.4 53.2 59.7 70.4
45.3 (1.5) 47 (1.5) 44.2 (1.8) 44.8 (1.6)
(2.2) (2.8) (2.2) (2.5)
13.5 (2.5) 6.0 (1.5) 7.1 (2.0) 33.9 (4.4) 27.6 (4.2) 16.8 (2.4)
0.00 0.00 0.25 0.00 0.00 0.73
(0.0) (0.0) (0.2) (0.0) (0.0) (0.4)
c2Z0.22; PZ0.64 t(44)ZK0.56; PZ0.57 t(44)Z1.63; PZ0.11 F (1/39)Z143.5; P!0.001 F (2/43)Z24.6; P!0.001 F (1/44)Z43.7; P!0.001 F (1/44)Z9.4; PZ0.004 F (9/36)Z12.45, P!0.001 F (1/44)Z27.5; P!0.001 F (1/44)Z3.45, PZ0.07 F (1/44)Z30.8, P!0.001 F (1/44)Z55.0, P!0.001 F (6/36)Z12.81, P!0.001 F (1/41)Z30.5, P!0.001 F (1/41)Z17.7, P!0.001 F (1/41)Z11.5, P!0.001 F (1/41)Z63.2, P!0.001 F (1/41)Z33.7, P!0.001 F (1/41)Z46.8, P!0.001
Mean (range). Mean (SEM).
subjects, mean age, and body mass index (BMI) did not differ significantly between the groups (Table 1). The mean duration of patients’ symptoms was 64.0 months, with a range from 17 to 168 months. Fourteen CFS patients reported an infectious onset of their symptoms. All patients reported an onset of symptoms within three months. The FS, HADS, SCL-90R and SIP scores of the CFS group were significantly higher than those of the control group and comparable to reported scores in previous studies (Table 1). One CFS patient fulfilled the criteria for a current Episode of Major Depression. However, since the exclusion of this subject did not alter the results of the analysis, the patient was included in the reported analysis. Seven patients reported a past history of major depression and four reported a past history of anxiety disorder. None of the controls reported any current or lifetime psychiatric disorder.
3.2. HPA axis responses in the TSST The TSST caused significant endocrine responses over time (ACTH: F (6/234)Z45.54; P!0.001; PC: F (6/234)Z39.57; P!0.001; SC: F (6/234)Z26.29; P!0.001). ANOVA revealed significant response differences over time between the groups for ACTH, but not for any cortisol parameter in the TSST (ACTH: F (1.69/65.9)Z3.47; PZ0.04; PC: F (2.59/101.06)Z1.19; PZ0.31; SC: F (2.5/99.74)Z
0.42; PZ0.70; Fig. 1). In addition, the area under the ACTH response curve was significantly reduced in patients compared to controls (F (1/39)Z6.34; PZ0.02;), but no differences in the cortisol parameters were observed (PC: F (1/39)Z0.1; PZ0.91; SC: F (1/39)Z1.03; PZ0.32; Fig. 1).
3.3. Immune responses in the TSST The total number of leukocytes increased significantly following psychosocial stress in both groups (time effect: F(1.36/46.24)Z3.923, PZ0.042). However, leukocyte numbers did not differ between patients and controls over time (group by time interaction effect: F(1.36/46.24)Z0.22, PZ 0.72; see Table 2, upper row). No stress-related changes in monocyte numbers or any group differences were observed (group: F(1/39)Z0.08, PZ 0.79; group by time interaction: F(1.54/52.61)Z 1.66, PZ0.20; see Table 2, lower row). The TSST caused significant group-specific responses in stimulated cytokine production (group by time interaction: Il-6: F (1.66/64.65)Z 4.00, PZ0.03, effect size f 2 Z0.17; TNF-a: F (1.71/66.81)Z4.75; PZ0.02; effect size f2Z 0.20). CFS patients showed an inverse response pattern in comparison to healthy controls, i.e. stimulated cytokine production decreased shortly after stress in CFS patients, while it increased in controls (Fig. 2). As glucocorticoids are known to
Inflammatory cytokines and CFS
193
Figure 1 ACTH, plasma cortisol and salivary cortisol responses over time and integrated endocrine responses (inserted bar graphs) of CFS patients (& or black) and healthy controls (C or gray) in the TSST.
exert an inhibitory influence on the secretion of pro-inflammatory cytokines, AUC of plasma and salivary free cortisol responses in the TSST were included as covariates in a subsequent ANCOVA for repeated measures. No differences in the level of significance and effect size measures were observed (PC: Il-6: F (1.63/62.82)Z3.93, PZ0.03,
effect size f2Z0.16; TNF-a: F (1.73/65.19)Z4.64; PZ0.02; effect size f2Z0.20; SC: Il-6: F (1.67/ 63.65)Z4.66, PZ0.02, effect size f2Z0.19; TNF-a: F (1.71/64.91)Z4.80; PZ0.02; effect size f2Z 0.20), indicating that absolute individual salivary free cortisol responses were not related to the differences in pro-inflammatory cytokine responses.
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Table 2
Immune responses in the TSST: changes of leukocyte and monocyte numbers after stress.
Parameter
Group
Samplea Basal
10 min after TSST
60 min after TSST
Leukocytes (!109 cells/l)
CFS Controls
5.98 (0.40) 5.85 (0.24)
6.10 (0.39) 5.91 (0.23)
6.17 (0.33) 6.16 (0.25)
Monocytes (!106 cells/l)
CFS Controls
451.82 (18.89) 478.20 (23.20)
480.36 (19.24) 456.83 (29.04)
444.40 (23.76) 491.38 (29.11)
a
Mean (SEM).
3.4. Associations between immune parameters and fatigue level To assess possible associations between stimulated pro-inflammatory cytokines levels and fatigue symptoms, Pearson’s correlations between the baseline (pre-TSST) as well as AUC of both cytokine responses curves and the scales of the FS were calculated for both groups separately (Table 3). For the control group, baseline levels of LPS-induced Il-6, but not TNF-a, were correlated with fatigue symptoms. The integrated cytokine responses in the TSST were not associated with fatigue levels in controls. In CFS patients, both baseline cytokine levels were significantly associated with fatigue scores. In addition, integrated cytokine responses in the TSST were significantly correlated with fatigue levels in the patients group. Correlation coefficients differed significantly between groups for baseline TNF-alpha and all integrated cytokine responses, but not for baseline Il-6 (data not shown). To examine whether significant correlations between AUC of cytokine responses and fatigue levels in CFS patients were a consequence of the existing association between baseline cytokine and fatigue levels, partial correlations between AUC of cytokine responses and fatigue levels controlling for baseline cytokine levels were performed. None of the correlations between AUC of cytokine responses and fatigue levels in CFS patients remained significant (data not shown).
response differences in cortisol parameters (Gaab et al., 2002a). In comparison to healthy controls, CFS patients were characterized by an inverted stimulated pro-inflammatory cytokine response during stress. Patients had comparable levels of LPS-induced Il-6 and TNF-a before and 60 min after the stress test, but unlike healthy controls, who showed a stress-induced increase of stimulated pro-inflammatory cytokine levels, CFS patients showed a decline in these parameters. The finding
4. Discussion This study set out to assess stimulated proinflammatory cytokine responses during psychosocial stress in CFS patients and healthy controls matched for age and gender. As previously reported, CFS patients had a significantly reduced ACTH response in the psychosocial stress test, which was not followed by similar
Figure 2 Pro-inflammatory cytokine responses of CFS patients (&) and healthy controls (C) in the TSST.
Inflammatory cytokines and CFS
195
Table 3 Pearson’s correlation between fatigue Scale total score and subscales and AUC of the stimulated proinflammatory cytokine responses in the TSST (significant correlations in bold). Baseline Il-6a
Baseline TNF-aa
AUC of Il-6a
AUC of TNF-aa
FS total score
CFS patients Control group
0.42, PZ0.03 0.45, PZ0.02
0.69, PZ0.000 0.12, PZ0.31
0.46, PZ0.02 0.18, PZ0.22
0.60, PZ0.002 0.16, PZ0.25
FS mental fatigue
CFS patients Control group
0.40, PZ0.04 0.49, PZ0.01
0.51, PZ0.001 0.15, PZ0.26
0.26, PZ0.13 0.16, PZ0.25
0.40, PZ0.04 0.16, PZ0.25
FS physical fatigue
CFS patients Control group
0.36, PZ0.05 0.39, PZ0.05
0.63, PZ0.001 0.08, PZ0.37
0.51, PZ0.01 0.19, PZ0.21
0.58, PZ0.003 0.16, PZ0.25
a
LPS-induced cytokine levels.
of short-term stress-induced increases in LPSstimulated pro-inflammatory cytokine levels in healthy subjects replicates the results of previous studies reporting increases of LPS-induced Il-6 and TNF-a after psychological or physical stress (Maes et al., 1998; Goebel et al., 2000). Groups did not differ with regard to total number of leukocytes and monocytes during and after the stress test. Furthermore, as indicated by the results of the ANCOVA, this inverse response pattern was not caused by possible differences in the individual absolute stress-induced cortisol responses. Calculating the associations between immune parameters and fatigue scores for each group separately, high correlations were found for baseline levels of Il-6 in both groups, whereas for baseline TNF-a, high correlations were found in CFS patients, but not in controls. Accordingly, only in CFS patients were integrated cytokine responses significantly correlated with fatigue symptoms. However, partial correlations revealed that these effects were a consequence of the high correlations between fatigue and cytokine levels at baseline. The results indicate that in the population under study, although cytokine responses were normal or attenuated, overall stimulated pro-inflammatory cytokine release is associated with general fatigue levels in CFS. However, it should be noted that this effect does not seem to be mediated through an exaggerated stress-induced increase in stimulated cytokine secretion. The observed cytokine response pattern does not appear to be caused by a possibly enhanced inhibition of cytokine production due to an exaggerated secretion of either plasma or salivary cortisol responses, since cortisol responses do not differ significantly between CFS patients and controls. With regard to inhibitory mechanisms of LPSinduced pro-inflammatory cytokine production, two important processes can be identified. On the one hand, glucocorticoids are known to inhibit the release of pro-inflammatory cytokines, thus promoting a shift from Th 1- to Th 2-dependent or cellular to humoral immune response, while
the effects of catecholamines are more heterogeneous. In-vitro, catecholamines suppress proinflammatory cytokines (Elenkov et al., 1996), while in-vivo, catecholamines may also activate cytokine production, for example via induction of NF-kB (Bierhaus, 2003, see below). It could be reasoned that the observed cytokine response differences are possibly not due to quantitative differences of the respective parameter, but rather to qualitative differences. From this point of view, it would not be the absolute amount that is important, but rather its relative effect. In CFS, a number of studies using different methods and investigating different target cells provide evidence for a general enhanced sensitivity to glucocorticoids, e.g. cytokine-specific supersensitivity of purified CD4 T cells to dexamethasone (Visser et al., 2001b), an enhanced sensitivity of pituitary corticotrophs to the inhibiting effects of 0.5 mg dexamethasone (Gaab et al., 2002b), an enhanced adrenal cortisol output to low but not to high levels of ACTH (Demitrack et al., 1991; Gaab et al., 2002a; Gaab et al., 2003b), and significant reduction of fatigue symptoms with treatment of 5–10 mg, but not 25–35 mg hydrocortisone (McKenzie et al., 1998; Cleare et al., 1999). In contrast, Kavelaars et al. (2000) observed a diminished capacity of dexamethasone to inhibit PHA-induced T cell proliferation, although the inhibitory concentration needed to achieve a 50% for DEX inhibition of PHA-induced cytokine production (IC50) was normal in CFS. This latter finding is in contrast to findings of enhanced glucocorticoid sensitivity for DEX inhibition of LPS-induced cytokine production, indicated by lower doses of DEX required for 50% inhibition of the maximum cytokine production observed after LPS stimulation without DEX (Gaab et al., 2003c), and an enhanced sensitivity of PHA-stimulated CD4 T cell and purified peripheral blood mononuclear cell proliferation to the suppressive effects of glucocorticoids (Visser et al.,1998, 2001b). These differences
196 could partly be explained by differences in the methods and subjects used, since Kavelaars et al. employed their methods in adolescent girls with CFS, while the contrasting studies were performed on adult patient populations. With regard to alteration of the sensitivity towards catecholamines, a reduced capacity of the b2-adrenergic agonist terbutaline to regulate the production of TNF-alpha and IL-10 has been reported in adolescent female CFS patients (Kavelaars et al., 2000). The exact mechanisms of the findings observed in our study and in the studies reported above remain to be elucidated, but given that the affinity, the number of GR, and the GR messenger RNA expression have been shown to be normal in CFS (Visser et al., 2001b), qualitative dysregulations of intracellular mechanism, e.g. the transduction of the signal from the receptor to the intracellular effector system, could be a possible candidate. The transcription factor nuclear factor (NF)-kB is a crucial signal for activation of the cellular inflammatory mechanism, e.g. cytokine production, adhesion molecule expression, etc. (McKay and Cidlowski, 1999). NF-kB is activated not only by cellular stress and inflammation signals, but also by psychosocial stress to which the whole organism is exposed, mediated via alpha-2 and beta-adrenergic receptors (Bierhaus et al., 2003). Glucocorticoids exert their inhibitory effects on cytokine production via suppression of NF-kB DNAbinding activity. Changes in the sensitivity of cells towards glucocorticoid inhibition can be mediated via a large array of extra- and intracellular mechanisms (Rohleder et al., 2003). In CFS patients, a decreased NF-kB activity in response to stress could be a possible intracellular mechanism to mediate the assumed increased glucocorticoid sensitivity. This would also be compatible with a decrease of cytokine production after stress. Attenuated catecholamine response to stress (which is unlikely, as this appears to be normal in CFS (Gaab et al., 2003a)), or lower sensitivity of cells towards catecholamines, for example due to a disturbed adrenoceptor balance (Kavelaars et al., 2000), could contribute to a decreased NF-kB activation by stress. However, these assumptions need to be tested in further studies. In addition, other mechanisms are also possible. With regard to the difference between patients and controls in the correlational pattern between LPS-induced pro-inflammatory cytokine secretion and fatigue levels, it needs to be noted that Il-6 has been shown to induce fatigue symptoms in healthy subjects and CFS patients (Arnold et al., 2002).
J. Gaab et al. However, in contrast to controls, CFS patients’ general fatigue levels were also strongly correlated with baseline LPS-induced TNF-a levels. From a somatic perspective, it has been suggested that cytokines, besides immediate effects, have protracted consequences through the promotion of behavioral and neurochemical sensitization (Anisman and Merali, 2003). Thus, behavioral responses to subsequent cytokine challenges are enhanced. For example, systemic TNF-a administration proactively enhanced the induction of sickness behavior to subsequent administration of the cytokine in mice (Hayley et al., 1999). In a recent study employing an Il-6 challenge to induce influenza-like symptoms, CFS patients experienced a greater amount of somatic symptoms with a shorter latency in comparison to healthy controls (Arnold et al., 2002). In CFS, stress, due either to psychiatric disorders, critical life events, or symptom-related distress, could lead to repeated secretion of pro-inflammatory cytokines, resulting in the previously described sensitization process. It has been suggested that “further increases in distress as a ‘reaction’ to mounting symptoms creates a vicious cycle” and that ’such a recursive system may act as a positive feedback loop, accounting for the chronic nature of CFS‘ (cited from: (Patarca, 2001), page 194). In conclusion, CFS patients did not show exaggerated pro-inflammatory cytokine responses during stress, but rather the opposite was the case. An enhanced sensitivity to the suppressive effects of glucocorticoids could serve as a possible physiological explanation for this finding. LPSinduced cytokine levels were strongly correlated with general fatigue levels in CFS patients, although cytokine levels were comparable to those of controls. An enhanced perception of cytokineinduced symptoms, possibly due to sensitization processes, could serve as an explanatory model to account for these findings. Further studies are needed to examine the role of stress-induced pro-inflammatory cytokine release in the perpetuation of the syndrome. For example, it would be interesting to evaluate the effects of interventions that aim to reduce stress and change symptom perception and attribution, such as cognitive behavioral therapy, on the correlation between pro-inflammatory cytokine secretion and CFS symptoms. In addition, it would be worth expanding the focus not only on pro-inflammatory cycles, but also on anti-inflammatory cytokines, since it has been shown that glucocorticoid regulation of pro- and anti-inflammatory cytokines is altered in CFS patients (Visser et al., 2000).
Inflammatory cytokines and CFS
References Anisman, H., Merali, Z., 2003. Cytokines, stress and depressive illness: brain-immune interactions. Ann. Med. 35, 2–11. Arnold, M.C., Papanicolaou, D.A., O’Grady, J.A., Lotsikas, A., Dale, J.K., Straus, S.E., Grafman, J., 2002. Using an interleukin-6 challenge to evaluate neuropsychological performance in chronic fatigue syndrome. Psychol. Med. 32, 1075–1089. Bierhaus, A., Wolf, J., Andrassy, M., Rohleder, N., Humpert, P.M., Petrov, D., Ferstl, R., von Eynatten, M., Wendt, T., Rudofsky, G., Joswig, M., Morcos, M., Schwaninger, M., McEwen, B., Kirschbaum, C., Nawroth, P.P., 20031. A mechanism converting psychosocial stress into mononuclear cell activation. Proc. Natl. Acad. Sci. USA 100, 1920–1925. Cannon, J.G., Angel, J.B., Abad, L.W., O’Grady, J., Lundgren, N., Fagioli, L., Komaroff, A.L., 1998. Hormonal influences on stress-induced neutrophil mobilization in health and chronic fatigue syndrome. J. Clin. Immunol. 18, 291–298. Chalder, T., Berelowitz, G., Pawlikowska, T., Watts, L., Wessely, S., Wright, D., Wallace, E.P., 1993. Development of a fatigue scale. J. Psychosom. Res. 37, 147–153. Clauw, D.J., Chrousos, G.P., 1997. Chronic pain and fatigue syndromes: overlapping clinical and neuroendocrine features and potential pathogenic mechanisms. Neuroimmunomodulation 4, 134–153. Cleare, A.J., 2003. The neuroendocrinology of chronic fatigue syndrome. Endocr. Rev. 24, 236–252. Cleare, A.J., Heap, E., Malhi, G.S., Wessely, S., O’Keane, V., Miell, J., 1999. Low-dose hydrocortisone in chronic fatigue syndrome: a randomised crossover trial. Lancet 353, 455–458. Cleeland, C.S., Bennett, G.J., Dantzer, R., Dougherty, P.M., Dunn, A.J., Meyers, C.A., Miller, A.H., Payne, R., Reuben, J.M., Wang, X.S., Lee, B.N., 2003. Are the symptoms of cancer and cancer treatment due to a shared biologic mechanism? A cytokine-immunologic model of cancer symptoms. Cancer 97, 2919–2925. Dantzer, R., 2001. Cytokine-induced sickness behavior: where do we stand?. Brain Behav. Immun. 15, 7–24. Demitrack, M.A., Dale, J.K., Straus, S.E., Laue, L., Listwak, S.J., Kruesi, M.J., Chrousos, G.P., Gold, P.W., 1991. Evidence for impaired activation of the hypothalamic-pituitary-adrenal axis in patients with chronic fatigue syndrome. J. Clin. Endocrinol. Metab. 73, 1224–1234. Dressendorfer, R.A., Kirschbaum, C., Rohde, W., Stahl, F., Strasburger, C.J., 1992. Synthesis of a cortisol-biotin conjugate and evaluation as a tracer in an immunoassay for salivary cortisol measurement. J. Steroid. Biochem. Mol. Biol. 43, 683–692. Elenkov, I.J., Papanicolaou, D.A., Wilder, R.L., Chrousos, G.P., 1996. Modulatory effects of glucocorticoids and catecholamines on human interleukin-12 and interleukin-10 production: clinical implications. Proc. Assoc. Am. Physicians 108, 374–381. Franke, G., 1995. Die Symptom-Checkliste von DerogatisDeutsche Version-Manual (2., rev. u. erw. Aufl.). Beltz Test GmbH, Weinheim. Fukuda, K., Straus, S.E., Hickie, I., Sharpe, M.C., Dobbins, J.G., Komaroff, A., 1994. The chronic fatigue syndrome: a comprehensive approach to its definition and study. International Chronic Fatigue Syndrome Study Group. Ann. Intern. Med. 121, 953–959.
197 Gaab, J., Huster, D., Peisen, R., Engert, V., Heitz, V., Schad, T., Schurmeyer, T.H., Ehlert, U., 2002a. Hypothalamic-pituitaryadrenal axis reactivity in chronic fatigue syndrome and health under psychological, physiological, and pharmacological stimulation. Psychosom. Med. 64, 951–962. Gaab, J., Huster, D., Peisen, R., Engert, V., Schad, T., Schurmeyer, T.H., Ehlert, U., 2002b. Low-dose dexamethasone suppression test in chronic fatigue syndrome and health. Psychosom. Med. 64, 311–318. Gaab, J., Engert, V., Schad, T., Heitz, V., Schu ¨rmeyer, T., Ehlert, U., 2003a. Catecholaminergic responsiveness under physiological stress in chronic fatigue syndrome. Psychosom. Med. 61, A26. Gaab, J., Huster, D., Peisen, R., Engert, V., Heitz, V., Schad, T., Schurmeyer, T., Ehlert, U., 2003b. Assessment of cortisol response with low-dose and high-dose ACTH in patients with chronic fatigue syndrome and healthy comparison subjects. Psychosomatics 44, 113–119. Gaab, J., Rohleder, N., Heitz, V., Engert, V., Schad, T., Schu ¨rmeyer, T., Ehlert, U., 2003c. Enhanced glucocorticoid sensitivity in patients with chronic fatigue syndrome. Acta Neuropsychiatr. 15, 184–191. Goebel, M.U., Mills, P.J., Irwin, M.R., Ziegler, M.G., 2000. Interleukin-6 and tumor necrosis factor-alpha production after acute psychological stress, exercise, and infused isoproterenol: differential effects and pathways. Psychosom. Med. 62, 591–598. Hayley, S., Brebner, K., Lacosta, S., Merali, Z., Anisman, H., 1999. Sensitization to the effects of tumor necrosis factoralpha: neuroendocrine, central monoamine, and behavioral variations. J. Neurosci. 19, 5654–5665. Herrmann, C., Buss, U., Snaith, R.P., 1995. HADS-D Hospital Anxiety and Depression Scale-Ein Fragebogen zur Erfassung von Angst und Depressivita ¨t in der somatischen Medizin. Hans Huber 1995. Kavelaars, A., Kuis, W., Knook, L., Sinnema, G., Heijnen, C.J., 2000. Disturbed neuroendocrine-immune interactions in chronic fatigue syndrome. J. Clin. Endocrinol. Metab. 85, 692–696. Kessler, S., Jaeckel, W., Cziske, R., 1997. Assessing health in musculoskeletal disorders–the appropriateness of a German version of the sickness impact profile. Rheumatol. Int. 17, 119–125. Kirschbaum, C., Pirke, K.M., Hellhammer, D.H., 1993. The ‘Trier Social Stress Test’—a tool for investigating psychobiological stress responses in a laboratory setting. Neuropsychobiology 28, 76–81. LaManca, J.J., Sisto, S.A., Zhou, X.D., Ottenweller, J.E., Cook, S., Peckerman, A., Zhang, Q., Denny, T.N., Gause, W.C., Natelson, B.H., 1999. Immunological response in chronic fatigue syndrome following a graded exercise test to exhaustion. J. Clin. Immunol. 19, 135–142. Lariviere, W.R., Melzack, R., 2000. The role of corticotropinreleasing factor in pain and analgesia. Pain 84, 1–12. Leese, G., Chattington, P., Fraser, W., Vora, J., Edwards, R., Williams, G., 1996. Short-term night-shift working mimics the pituitary-adrenocortical dysfunction in chronic fatigue syndrome. J. Clin. Endocrinol. Metab. 81, 1867– 1870. Lloyd, A., Gandevia, S., Brockman, A., Hales, J., Wakefield, D., 1994. Cytokine production and fatigue in patients with chronic fatigue syndrome and healthy control subjects in response to exercise. Clin. Infect. Dis. 18, S142–S146. Luger, A., Deuster, P.A., Kyle, S.B., Gallucci, W.T., Montgomery, L.C., Gold, P.W., Loriaux, D.L., Chrousos, G.P.,
198 1987. Acute hypothalamic-pituitary-adrenal responses to the stress of treadmill exercise. Physiologic adaptations to physical training. N. Engl. J. Med. 316, 1309–1315. Lutgendorf, S.K., Antoni, M.H., Ironson, G., Fletcher, M.A., Penedo, F., Baum, A., Schneiderman, N., Klimas, N., 1995. Physical symptoms of chronic fatigue syndrome are exacerbated by the stress of Hurricane Andrew. Psychosom. Med. 57, 310–323. Maes, M., Song, C., Lin, A., De Jongh, R., Van Gastel, A., Kenis, G., Bosmans, E., De Meester, I., Benoy, I., Neels, H., Demedts, P., Janca, A., Scharpe, S., Smith, R.S., 1998. The effects of psychological stress on humans: increased production of proinflammatory cytokines and a Th1-like response in stressinduced anxiety. Cytokine 10, 313–318. McEwen, B.S., Biron, C.A., Brunson, K.W., Bulloch, K., Chambers, W.H., Dhabhar, F.S., Goldfarb, R.H., Kitson, R.P., Miller, A.H., Spencer, R.L., Weiss, J.M., 1997. The role of adrenocorticoids as modulators of immune function in health and disease: neural, endocrine and immune interactions. Brain Res. Brain Res. Rev. 23, 79–133. McKay, L.I., Cidlowski, J.A., 1999. Molecular control of immune/inflammatory responses: interactions between nuclear factor-kappa B and steroid receptor-signaling pathways. Endocr. Rev. 20, 435–459. McKenzie, R., O’Fallon, A., Dale, J., Demitrack, M., Sharma, G., Deloria, M., Garcia-Borreguero, D., Blackwelder, W., Straus, S.E., 1998. Low-dose hydrocortisone for treatment of chronic fatigue syndrome: a randomized controlled trial. J. Am. Med. Assoc. 280, 1061–1066. Natelson, B.H., 2001. Chronic fatigue syndrome. J. Am. Med. Assoc. 285, 2557–2559. Papanicolaou, D.A., Petrides, J.S., Tsigos, C., Bina, S., Kalogeras, K.T., Wilder, R., Gold, P.W., Deuster, P.A., Chrousos, G.P., 1996a. Exercise stimulates interleukin-6 secretion: inhibition by glucocorticoids and correlation with catecholamines. Am. J. Physiol. 271, E601–E605. Papanicolaou, D.A., Tsigos, C., Oldfield, E.H., Chrousos, G.P., 1996b. Acute glucocorticoid deficiency is associated with plasma elevations of interleukin-6: does the latter participate in the symptomatology of the steroid withdrawal syndrome and adrenal insufficiency?. J. Clin. Endocrinol. Metab. 81, 2303–2306. Patarca, R., 2001. Cytokines and chronic fatigue syndrome. Ann. N Y Acad. Sci. 933, 185–200. Peterson, P.K., Sirr, S.A., Grammith, F.C., Schenck, C.H., Pheley, A.M., Hu, S., Chao, C.C., 1994. Effects of mild exercise on cytokines and cerebral blood flow in chronic fatigue syndrome patients. Clin. Diagn. Lab. Immunol. 1, 222–226. Pruessner, J.C., Kirschbaum, C., Meinlschmid, G., Hellhammer, D.H., 2003. Two formulas for computation of the area under the curve represent measures of total hormone concentration versus time-dependent change. Psychoneuroendocrinology 28, 916–931.
J. Gaab et al. Rohleder, N., Wolf, J.M., Kirschbaum, C., 2003. Glucocorticoid sensitivity in humans-interindividual differences and acute stress effects. Stress 6, 207–222. Sharpe, M.C., Archard, L.C., Banatvala, J.E., Borysiewicz, L.K., Clare, A.W., David, A., Edwards, R.H., Hawton, K.E., Lambert, H.P., Lane, R.J., et al., 1991. A report—chronic fatigue syndrome: guidelines for research. J.R. Soc. Med. 84, 118–121. Steptoe, A., Willemsen, G., Owen, N., Flower, L., MohamedAli, V., 2001. Acute mental stress elicits delayed increases in circulating inflammatory cytokine levels. Clin. Sci. (Lond) 101, 185–192. Theorell, T., Blomkvist, V., Lindh, G., Evengard, B., 1999. Critical life events, infections, and symptoms during the year preceding chronic fatigue syndrome (CFS): an examination of CFS patients and subjects with a nonspecific life crisis. Psychosom. Med. 61, 304–310. Vgontzas, A.N., Zoumakis, M., Papanicolaou, D.A., Bixler, E.O., Prolo, P., Lin, H.M., Vela-Bueno, A., Kales, A., Chrousos, G.P., 2002. Chronic insomnia is associated with a shift of interleukin-6 and tumor necrosis factor secretion from nighttime to daytime. Metabolism 51, 887–892. Visser, J., Blauw, B., Hinloopen, B., Brommer, E., de Kloet, E.R., Kluft, C., Nagelkerken, L., 1998. CD4 T lymphocytes from patients with chronic fatigue syndrome have decreased interferon-gamma production and increased sensitivity to dexamethasone. J. Infect. Dis. 177, 451–454. Visser, J., Graffelman, W., Blauw, B., Haspels, I., Lentjes, E., de Kloet, E.R., Nagelkerken, L., 2001a. LPS-induced IL-10 production in whole blood cultures from chronic fatigue syndrome patients is increased but supersensitive to inhibition by dexamethasone. J. Neuroimmunol. 119, 343–349. Visser, J., Lentjes, E., Haspels, I., Graffelman, W., Blauw, B., de Kloet, R., Nagelkerken, L., 2001b. Increased sensitivity to glucocorticoids in peripheral blood mononuclear cells of chronic fatigue syndrome patients, without evidence for altered density or affinity of glucocorticoid receptors. J. Investig. Med. 49, 195–204. Visser, J.T., De Kloet, E.R., Nagelkerken, L., 2000. Altered glucocorticoid regulation of the immune response in the chronic fatigue syndrome. Ann. NY Acad. Sci. 917, 868–875. Vollmer-Conna, U., Lloyd, A., Hickie, I., Wakefield, D., 1998. Chronic fatigue syndrome: an immunological perspective. Aust. NZJ Psychiatr. 32, 523–527. Wittchen, H.-U., Pfister, H., 1997. DIA-X Interviews: Manual fu ¨r Screening-Verfahren und Interview. Swets and Zeitlinger, Frankfurt. Wood, G.C., Bentall, R.P., Gopfert, M., Dewey, M.E., Edwards, R.H., 1994. The differential response of chronic fatigue, neurotic and muscular dystrophy patients to experimental psychological stress. Psychol. Med. 24, 357–364.