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Departments of Neurology (L.M.L.v.W., C.H.P., B.M.J.U.), Molecular Cell Biology (D.F.R.M., C.D.D., T.K.v.d.B.), and Clinical Epidemiology and Biostatistics (B.M.J.U.), Vrije Universiteit Medical Center, 1007 MB Amsterdam, The Netherlands
Address all correspondence and requests for reprints to: L. van Winsen, Vrije Universiteit Medical Center, Department of Neurology, P.O. Box 7057, 1007 MB Amsterdam, The Netherlands. E-mail: l.vanwinsen2{at}vumc.nl.
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Abstract |
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production. Blood cells of MS patients, especially relapsing remitting MS patients, were less sensitive to GC compared with blood cells of healthy controls. This turned out to be unrelated to previous treatment with exogenous GC expressed as frequency of courses of iv steroids or interval since last course. The use of interferon ß was found to be associated with a lower GC sensitivity. However, after correction for the use of interferon ß, relapsing remitting MS patients remained less sensitive to GC. |
Introduction |
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The production of cortisol is regulated by the hypothalamic-pituitary-adrenal (HPA) axis. An increased activity of the HPA axis leads to an increased production of cortisol. Cortisol exerts its action via interaction with GC receptors that are present in nearly every cell in the human body. Because of a negative feedback system mediated via the GC receptors, elevated levels of cortisol will usually lead to a decrease in HPA axis activity (3).
In subgroups of MS patients, this feedback system seems to be disturbed. Signs of increased basal secretion of cortisol have been found (5, 6). In postmortem studies in MS patients, an increased size of adrenal glands was observed (7). Other postmortem studies revealed higher numbers (8) and activity of hypothalamic neurons producing CRH (9). The clinical relevance of an increased HPA axis activity is supported by the observation that this phenomenon is related to the clinical disease course (10). Moreover, a recent study revealed that the HPA axis may have a modulatory effect on inflammatory disease activity in MS. High HPA axis activity was associated with lower amount of gadolinium-enhanced lesions on magnetic resonance imaging (MRI), suggesting a protective effect of increased HPA axis activity (11).
The mechanism behind increased HPA axis activity in MS is unknown, but it may be a result of a reduced negative feedback mechanism. Functional studies showed a decreased response to the dexamethasone (DEX) suppression test (12) and increased cortisol levels after combined DEX suppression-CRH tests (10, 13, 14, 15, 16). In depressive patients, increased HPA axis activity was associated with decreased peripheral GC sensitivity (17). Despite elevated cortisol levels, clinical signs of hypercortisolism in MS are unusual. Taken together, these observations suggest that increased HPA axis activity is accompanied by a reduction in the GC sensitivity (16). Some older studies show conflicting results with respect to GC sensitivity in MS (18, 19, 20). A small-scale study in 19 MS patients did not show differences in GC sensitivity between MS patients and healthy controls (HCs) or between patients with and without gadolinium enhancement on MRI (21). However, a recent study in 24 patients showed a reduced GC sensitivity in relapsing remitting (RR) MS patients (22).
To evaluate the hypothesis that peripheral GC sensitivity is decreased in MS patients compared with HCs, we have set up a larger study. In addition, we examined the relationship between GC sensitivity and clinical disease characteristics. GC sensitivity was determined using the suppressive effect of GC on in vitro-stimulated cytokine production. In our analyses, the possible effect of prior treatment with corticosteroids on GC sensitivity was taken into account because it has been shown that binding of GC to the GC receptor (GR) results in a significant down-regulation of the expression of the GR (23). Based on preliminary observations that interferon ß (IFN-ß), a current frequently used drug in MS, can influence GC sensitivity (24, 25), we were also interested in the effect of in vivo treatment with IFN-ß on GC sensitivity.
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Patients and Methods |
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Whole blood was obtained from 126 MS patients and 51 healthy volunteers. All patients visited the outpatient clinic and met the Poser criteria for clinically definite MS (26). Patient files were scrutinized to obtain data on all prior exposure to corticosteroids. The volunteers were recruited mainly from personnel working in the hospital or the laboratory. The study was approved by the ethics committee of the Vrÿe Universiteit Medical Centre (Amsterdam, The Netherlands), and all patients and healthy volunteers gave informed consent.
Whole-blood cultures
The whole-blood stimulation procedure was based on a previously described method (27) with some minor modifications. Briefly, blood was collected by venapuncture and collected in chromogenix endotoxin-free, heparinized tubes (CN Smith, Chromogeneix Endotube, Molndal, Sweden). To limit the influence of circadian variation in cortisol levels and cytokine production (28), blood was obtained between 0900 and 1100 h. The blood was diluted 1:1 in RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 0.01% L-Glutamine 200 nM (GIBCO). All experiments were performed in 96-well tissue culture plates (Greiner, Frickenhausen, Germany). Cultures were set up in triplicate with each well containing 200 µl of diluted whole blood. During pipetting, the cell suspension was frequently vortexed to prevent sedimentation. To induce TNF- production, cells were stimulated with lipopolysaccharide (LPS; Escherichia coli; final concentration, 100 ng/ml) except for a triple control for spontaneous TNF- production. A dose-related response to DEX (Sigma, St. Louis, MO) was studied by the addition of DEX in a final concentration ranging from 10–10-10–5 M. One stock solution of 10–2 M was prepared in ethanol and stored at –80 C in 150-µl aliquots. The supernatants of the cultures were collected after 4 h of incubation at 37 C and stored at –20 C pending TNF- measurements.
Cytokine measurements
TNF- in the cell culture supernatants was measured with a commercial ELISA (Biosource, Nivelles, Belgium) using MaxiSorp surface 96-well plates (NUNC, Roskilde, Denmark). Recombinant TNF- was diluted in culture medium and used as a standard. The standard curves ranged from 4–1000 pg/ml.
IC50 calculation
IC50 was determined as a log concentration of DEX causing 50% of the maximum inhibitory effect. Inhibition of TNF- by DEX was expressed as a percentage of LPS-induced TNF- in the absence of DEX. To calculate the IC50 for each subject, a sigmoid dose-response curve for inhibition by DEX was fitted using the following tangens hyperbolicus function: TNF- = a * tangens hyperbolicus [b * log concentration (DEX – c)] + d (Fig. 1
, A–G). As a result of this calculation, IC50 takes into account the maximum TNF- value after stimulation with LPS and the suppressive effect of different concentrations of DEX, ranging from very low concentrations to the maximum achievable suppression at high concentrations of DEX (29).
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values outside the linear area of the standard curve. Statistical analysis
Mean values of IC50 between patients and HCs were compared using the independent Student’s t test, with a two-tailed significance set at 0.05. MS subtypes and HCs were compared by ANOVA, and Bonferroni’s post hoc correction for multiple tests was used to compare MS subtypes and HCs. In a subsequently performed ANOVA, the use of IFN-ß was included as a covariate. To investigate the contribution of a predefined set of patient and clinical disease characteristics on the IC50, multiple linear regression analysis was undertaken. Cox regression analysis was performed to study the effect of the IC50 on disease progression as defined by time to Expanded Disability Status Scale (EDSS) 6.
The level of significance was set at 0.05. All statistical analyses were performed using SPSS for windows, version 11.0 (SPSS, Chicago, IL).
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Results |
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The group of MS patients consisted of 74 patients with RR MS, 35 patients with secondary progressive (SP) MS, and 17 patients with primary progressive (PP) MS (31). Of the RR and SP MS patients, 38 were on IFN-ß treatment. Seventy-four patients had previously been exposed to systemic corticosteroids. In all cases, this systemic treatment of corticosteroids consisted of a course of high-dose iv methylprednisolone (iv-MP), given in a dose of either 1000 mg for 3 consecutive days or as 500 mg for 5 consecutive days. The number of iv-MP treatments ranged from 1–15, and the minimum interval between last exposure to corticosteroids and blood withdrawal was 30 d. In none of the subjects was there any detectable spontaneous secretion of TNF- (i.e. in the absence of LPS, levels of TNF- were below the level of assay sensitivity).
In 15 subjects, TNF- production after stimulation with LPS was outside the linear area of the standard curve, and therefore, these subjects, consisting of six HCs (12%) and nine MS patients (7%), were not included in the analysis. Characteristics of patients and HCs that were included in the analysis are shown in Table 1. Characteristics of RR and SP patients with and without IFN-ß treatment are shown in Table 2 and are comparable except with respect to previous exposure to iv-MP; more patients on IFN-ß treatment had been exposed to iv-MP compared with patients without IFN-ß treatment, and the number of courses was higher as well (P < 0.01). Among all patients treated with iv-MP in the past, the clinical effect of iv-MP was not any different between patients who were not treated vs. patients who were treated with IFN-ß. If anything, the treatment effect in the latter group was even better (P = 0.037, not corrected for multiple comparisons). In patients who were treated with iv-MP both before and after onset of IFN-ß treatment, the clinical effects in these two periods were comparable.
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Blood cells of MS patients were less sensitive to GC compared with HCs (P < 0.001). In HCs, the mean IC50 was 23.7 nM, and, in MS patients, the mean IC50 was 35.4 nM (95% confidence interval of the difference, which was 11.7 nM, is 5.6–17.8 nM). ANOVA revealed a difference between clinical MS subtypes with respect to the IC50 (Fig. 2). Bonferroni’s post hoc test showed that this difference was due to a difference between HCs vs. RR MS patients (P = 0.001) and HCs vs. SP MS patients (P = 0.008), whereas there was no difference between HCs and PP MS patients. Within the MS patients, the differences between the several subgroups were not significant (RR vs. PP, P = 0.23; SP vs. PP, P = 0.28; and RR vs. SP, P = 1.0).
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In a multiple linear regression analysis in which the IC50 was used as the dependent variable and as independent variables were entered (gender, age, MS subtype, EDSS score, disease duration, IFN-ß treatment, number of iv-MP treatments in the past, time in months since last iv-MP treatment, whether patients were in an active or stable disease phase, and season of blood sampling), only IFN-ß treatment significantly contributed to the IC50 (ß = 0.254, P < 0.01). Patients on IFN-ß treatment had a lower GC sensitivity. None of the other parameters were shown to have an effect on GC sensitivity.
After excluding patients on IFN-ß treatment in the ANOVA analysis with Bonferroni correction, the difference in IC50 between HCs and MS patients remained significant (P = 0.003), but only RR MS remained significantly less sensitive compared with HCs (P = 0.021), as illustrated in Fig. 3.
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Effects of exogenous GC on IC50
Mean IC50 for patients with or without iv-MP treatment in the past was 36.2 nM (SD, 19.7 nM) and 34.1 nM (SD, 18.4 nM), respectively. MS patients who had never been treated with iv-MP were less sensitive compared with HCs (mean IC50 for HCs, 23.7 nM, P = 0.003; 95% confidence interval of the difference, –17.1 to –3.6 nM; Fig. 4). Patient characteristics show that patients who had never been treated with GC were mainly RR MS patients who had lower EDSS scores and who were less often on IFN-ß therapy (Table 3).
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Discussion |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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In other inflammatory or autoimmune diseases such as rheumatoid arthritis (32), asthma (33), Crohn’s disease (34), and ulcerative colitis (35, 36), evidence of decreased GC sensitivity of blood cells has been reported. The underlying mechanism leading to this reduction in GC sensitivity is unknown. It has often been suggested that a possible explanation for this reduced GC sensitivity might be the frequent administration of exogenous GC, which may be the case in some of these diseases. However, in this study, we applied multiple methods to analyze this possible correlation and were unable to identify any relationship between decreased GC sensitivity and either number of courses of iv-MP or interval since last course. In fact, even after excluding all patients who had ever been treated with exogenous GC, MS patients were still less sensitive to GC compared with controls.
Until now, studies evaluating peripheral GC sensitivity in MS patients showed conflicting results (18, 19, 20, 21, 22). In most of these studies, GC sensitivity was measured as suppressive effect of DEX on phytohemagglutinin-induced mitogenic response of isolated peripheral blood mononuclear cells. In our study, GC sensitivity was measured as suppressive effect of DEX on LPS-stimulated TNF- production, which is predominantly monocyte driven (37). Using whole blood preserves the natural environment (including endogenous cortisol) of cytokine-producing cells, which will at least partially be influenced by the HPA axis (3, 4). Our results are in line with a recent study in which the suppressive effect of DEX on IL-6 after whole-blood LPS stimulation was evaluated (22). In this study, however, no clear distinction between PP and SP MS was made. Furthermore, our study is based on, as of yet, the largest sample. Intraindividual variation of IC50 has been investigated in lymphocytes in HCs and was as great as between-individual variation (29). Other studies showed that GC sensitivity of lymphocytes in MS patients remained constant over time (18, 21). All these studies have been done in isolated PBMCs in which there is more intraindividual variability regarding cytokine production (28).
In MS, HPA axis dysregulation has been described, but the underlying mechanism is still unclear. HPA axis dysregulation was presumed to be due to diminished corticoid receptor function (16). Apart from being caused by a decreased peripheral GC sensitivity, a chronic activation of the HPA axis may result in a down-regulation of the peripheral GC sensitivity to protect tissue from increased cortisol levels, as is the case in patients with the GC resistance syndrome and in endogenous Cushing’s syndrome, although this protecting mechanism seems to be insufficient in patients with the latter because they do develop clinical signs of GC excess (38). Also in MS, an increased HPA axis activity is likely to go along with a decreased peripheral GC sensitivity because clinical signs of hypercortisolism are unusual. However, HPA axis activity was not formally addressed in this study. Moreover, the activity of the HPA axis may be independent from the responsiveness to acutely exogenous administered GC (39).
There are several possible explanations for a change in GC sensitivity, but these, thus far, have not been studied in MS. Different ratios between the receptor isoform GR- and GR-ß, an alternatively spliced variant of GR- that does not bind to GC but antagonizes the transactivating activity of the classic GR- receptor (40), have been described as possible explanations for differences in GC sensitivity in asthma (41) and ulcerative colitis (42). Decreased GC sensitivity may also be genetically determined. Several polymorphisms in the GR have been described. Some have been associated with altered GC sensitivity in the primary GC resistance syndrome, but have also been observed in HCs (43). It will be interesting to investigate the role of these polymorphisms in MS.
In this study, we evaluated the role of several parameters that could have confounded our observations. First, we looked at previous treatments with GC. In the MS patients studied here, prior iv-MP treatment, number of iv-MP in history, and months since last iv-MP therapy did not influence IC50. Therefore, our finding that RR MS and SP MS patients have decreased GC sensitivity compared with HCs and PP MS patients cannot be explained by previous iv-MP treatment. Even in analyses excluding all patients who had ever received exogenous GC, RR MS patients were still less sensitive to GC compared with HCs. Second, we looked at disease activity because acute inflammation may influence IC50. Unfortunately, MRI data were not available for all patients at the time of data collection. Therefore, we could only rely on clinical observations with respect to disease activity. We did not observe differences between patients in a clinically active phase of the disease vs. those in a stable phase. This could be due to the relatively small number of patients in active disease (n = 26), or it may suggest that GC sensitivity, as measured in this study, is a relative constant factor changing under influence of chronic inflammation rather than during acute inflammation. Third, seasonal variation in GC activity has been described (44) in healthy men and in MRI and immune markers in MS patients (45). We compared IC50 in the four seasons, which were determined as spring (March, April, and May), summer (June, July, and August), autumn (September, October, and November), and winter (December, January, and February), and no significant differences were found.
Finally, the use of IFN-ß can influence the HPA axis, resulting in increased endogenous cortisol levels, as was shown in patients with cancer (46) and chronic hepatitis (47), as well as in HCs (48). One small study in 10 MS patients did not establish an effect of 4–6 months of IFN-ß treatment on HPA axis parameters (49). In our study, IFN-ß treatment was associated with lower GC sensitivity. IFN-ß treatment is especially initiated in patients with clinically active disease, which is probably caused by an increased and ongoing inflammation in the central nervous system. This may be related to or, via a defective suppression by endogenous GC, even caused by a decreased GC sensitivity. The observed effect of IFN-ß should perhaps not be contributed to IFN-ß itself but rather explained by selection bias. When carefully comparing the disease characteristics of our RR and SP patients, we observed a significant difference in exposure to iv-MP treatment, thereby supporting the latter explanation.
The aim of our study was to gain more insight in the presence and role of peripheral GC sensitivity in MS patients compared with HCs. MS patients, especially RR MS patients, were less sensitive to GC. It is tempting to speculate that this is related to a defective mechanism in controlling inflammation in MS patients. Further studies to elucidate the implications and causes of a reduced GC sensitivity in MS are warranted.
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Acknowledgments |
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Footnotes |
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Abbreviations: DEX, Dexamethasone; EDSS, Expanded Disability Status Scale; GC, glucocorticoid; GR, GC receptor; HC, healthy control; HPA, hypothalamic-pituitary-adrenal; IFN-ß, interferon ß; iv-MP, iv methylprednisolone; LPS, lipopolysaccharide; MRI, magnetic resonance imaging; MS, multiple sclerosis; PP, primary progressive; RR, relapsing remitting; SP, secondary progressive.
Received February 17, 2004.
Accepted October 27, 2004.
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References |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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production by increasing the number of glucocorticoid receptors on a human monocytic cell line. J Interferon Cytokine Res 21:133–135[CrossRef][Medline]
production in whole blood. J Leukoc Biol 52:687–692[Abstract]
, IL-6, IL-2, IFN-
and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation. Cytokine 4:239–248[CrossRef][Medline]
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) of IC50 (nM) of HCs and MS subtypes (ANOVA, P < 0.001; Bonferroni’s post hoc test: RR MS vs. PP MS, P = 0.32; SP MS vs. PP MS, P = 0.41; HCs vs. RR MS, P = 0.001; HCs vs. SP MS, P = 0.008; and HCs vs. PP MS, P = 1.0).
