Corticosteroid resistance in a subpopulation of multiple sclerosis patients as measured by ex vivo dexamethasone inhibition of LPS induced IL-6 production

Journal of Neuroimmunology 151 (2004) 180 – 188 www.elsevier.com/locate/jneuroim

Corticosteroid resistance in a subpopulation of multiple sclerosis patients as measured by ex vivo dexamethasone inhibition of LPS induced IL-6 production Roel H. DeRijk *, Farideh Eskandari, Esther M. Sternberg Section in Neuroendocrine Immunology and Behavior, National Institute of Mental Health, 36 Convent Drive (MSC 4020), Bethesda, MD 20892-4020, USA Received 22 October 2003; received in revised form 19 February 2004; accepted 20 February 2004

Abstract We assessed corticosteroid sensitivity in multiple sclerosis (MS) patients compared to control subjects, using an in vitro assay of dexamethasone (Dex) inhibition of lipopolysaccharide (LPS) stimulated-blood interleukin-6 production. Significantly higher concentrations of dexamethasone were needed to obtain 50%-inhibition (ID50) of in vitro LPS stimulated interleukin (IL)-6 production (28.4  10 7 M) in relapsing – remitting MS (RRMS) patients compared to chronic progressive MS (CPMS) patients (6.2  10 7 M) or compared to controls (3.0  10 7 M). We also found a trend towards worsening of clinical status over time with increasing corticosteroid resistance. These data suggest that corticosteroid sensitivity may be a factor in the pathogenesis and could be used for prognosis of MS. D 2004 Published by Elsevier B.V. Keywords: Multiple sclerosis; Cortisol; Corticosteroid sensitivity; IL-6 production; Dexamethasone

1. Introduction Susceptibility to inflammatory and immune illnesses is influenced by the activity of the hypothalamic-pituitaryadrenal (HPA) axis (Eskandari and Sternberg, 2002; Derijk and Sternberg, 1994) as shown in animal models (Sternberg et al., 1989; Dietrich et al., 1997; Lechner et al., 1996) and human autoimmune diseases (Buske-Kirschbaum et al., 2002; Straub et al., 2002; Demitrack and Crofford, 1998; Johnson et al., 1998; Gutierrez et al., 1998, 1999; Michelson et al., 1994; Chikanza et al., 1992; Neek et al., 2002). Stimulation of the HPA axis by cytokines results in elevated circulating levels of corticosterone, which in turn modulate cytokine patterns and suppress inflammation through a negative feedback loop. After administration of myelin basic protein, LEW/N rats develop experimental allergic encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). By contrast, F344/N rats, a strain closely related to LEW/N rats but with a much more robust * Corresponding author. Gezondheidscentrum Rijngeestgroep, Psychiatric Hospital, Oegstsgeest, Endegeesterstraatweg 5, 2342 AJ Oegstgeest, The Netherlands. Tel.: +31-71-8907059. E-mail address: [email protected] (R.H. DeRijk). 0165-5728/$ - see front matter D 2004 Published by Elsevier B.V. doi:10.1016/j.jneuroim.2004.02.009

corticosteroid response, are relatively resistant to the induction of EAE and other autoimmune and inflammatory diseases. Moreover, EAE can be prevented by the administration of physiological dosages of corticosterone, whereas EAE-resistant strains are made susceptible by adrenalectomy (Mason et al., 1990). In these studies, increasing doses of replacement steroids were inversely related to disease susceptibility. These data indicate the importance of appropriate HPA axis functioning as a crucial factor determining susceptibility and resistance to and severity of EAE disease expression. Several lines of evidence suggest that in human MS, the HPA axis is also dysregulated. We have shown that MS patients have significantly higher basal levels of circulating plasma cortisol, and an altered regulation of the HPA axis (Michelson et al., 1994), although no differences in cortisol output following stimulation of the pituitary was observed compared to healthy controls. It can be argued that a decreased cortisol feedback at the level of the hippocampus could produce these changes and could account for the increased amount and activity of corticotropin releasing hormone (CRH) in the paraventricular nucleus in MS patients (Erkut et al., 1995). Consistent with this notion is the finding that up to 50% of MS patients studied failed to

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suppress cortisol appropriately after the administration of dexamethasone (Dex) (Reder et al., 1987), while adrenal size is increased in these patients (Reder et al., 1994). In addition, using a combined Dex-suppression and CRHstimulation test, MS patients show higher cortisol responses than healthy controls, suggesting the possibility of corticosteroid resistance (Grasser et al., 1996). Finally, the need to administer high pharmacological doses of synthetic corticosteroids to suppress disease exacerbations also points to decreased corticosteroid sensitivity in inflamed tissue (van Oosten et al., 1995). Thus, considering the crucial role of cortisol in immune regulation, a reduced tissue-sensitivity to cortisol could account for altered cortisol responses in MS (DeRijk et al., 1997b). In this study, we examined corticosteroid sensitivity in MS patients using in vitro Dex inhibition of lipopolysaccharide (LPS)-induced interleukin (IL)-6 responses in whole blood. Induction of cytokine production in whole blood is now widely used and has the advantage that the cells are in a relatively natural environment, since no isolation steps are used (Nerad et al., 1992). Moreover, absence of isolation procedures could be a crucial step in preserving the original corticosteroid sensitivity in vitro, since corticosteroid sensitivity has now been shown to be highly dynamic (DeRijk et al., 1996; Ebrecht et al., 2000; Rohleder et al., 2003; Duclos et al., 2003). We chose IL-6 production as the cytokine readout because, in our hands it was found to be insensitive to circadian variations in plasma cortisol (DeRijk et al., 1997a). IL-6 has recently been implicated in MS, not only because it is a central regulator of the acute phase response, including immune responses, fever and HPA axis activation (Akira et al., 1990), but also because IL-6 deficient mice fail to develop EAE (Mendel et al., 1998; Eugster et al., 1998; Samoilova et al., 1998). Furthermore, changes in IL-6 expression in human MS lesions or other component of the IL-6 system have been reported (Padberg et al., 1999; Schonrock et al., 2000). Finally, patterns of pro-inflammatory monokines, including IL-6, may be used to differentiate between subgroups of MS patients (Kleine et al., 2003). We also examined the correlation of clinical status of MS patients with corticosteroid sensitivity. We used the expanded disability status scale (EDSS) score as an indicator of severity of the MS clinical status, higher score indicating a greater degree of disability/lower functional status.

2. Materials and methods 2.1. Patients Patients were recruited from the MS clinic at the National Institutes of Health (NIH, Bethesda, MD). The study was approved by the National Institute of Neurological Disease and Stroke (NINDS) institutional review board. All patients were seen by a neurologist (H. McF.) to confirm the diagnosis of MS using the Poser criteria and the severity

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of illness using the EDSS. Samples were collected over a period of 2 years. The average age (mean F S.D.) of the participants was not significantly different: healthy controls 38 F 8 years, relapsing – remitting MS (RRMS) group 37 F 7 years and chronic progressive MS (CPMS) group 43 F 7 years. In the healthy control group (n = 12), none of the subjects was treated with anti-inflammatory drugs. Concurrent or previous medication use is shown in Table 1. In the RRMS group (n = 13), five subjects had received no anti-inflammatory drugs at all; two had received betaserone (interferon h-1b) in combination with azathioprine or cyclosporin; five had received i.v. prednisolone, one 3 weeks before blood collection, one 6 weeks before blood collection along with betaserone, one 10 weeks before blood collection and two 12 weeks before blood collection. One patient had not received high dose steroids in the 3 months prior to the study, but no data was available prior to that time regarding drug therapy in this subject. In the CPMS group (n = 11), four had not received any anti-inflammatory drugs; one was treated with betaserone; one was treated with 15-deoxyspergualine (DSG); while i.v. prednisolone was given to three patients, one 6 weeks before blood collection, one 7 weeks before blood collection and one 8 weeks before blood collection. Two patients had not received high dose steroids in the 3 months prior to the study, but no data was available prior to that time regarding drug therapy in these two subjects. 2.2. Ex vivo stimulation of whole blood Venous blood was collected in heparinized tubes (15 IU/ ml blood, sodium heparin, 8 ml tubes, No. 6541, Becton and

Table 1 Clinical data and Dex ID50 of subjects Controls

CPMS

RRMS

Age/ ID50 Age/ ID50 sex sex

Treatment

Age/ ID50 sex

1

49/M 8.0

54/M 30.0

No

2 3

30/M 6.0 40/M 3.1

29/M 15.0 42/M 4.5

Pred, 8 Wks DSG, 9 Mts

4 5

38/M 3.0 33/M 2.8

39/F 55/M

4.0 3.0

Pred, 7 Wks No>3 Mts

6 7 8 9 10 11 12 13

49/M 46/M 27/F 37/F 26/M 35/M 45/F

41/F 39/F 46/M 41/F 47/M 43/F

2.9 2.9 2.6 1.7 1.2 0.45

No No Betaseron Pred, 6 Wks No>3 Mts No

41/M 100.0 Pred, 6 Wks, Betaseron 49/F 79.0 No 31/M 60.0 Betaseron, Cyclosporin 33/M 30.0 No 29/F 22.0 Betaseron, Azatioprine 42/M 21.0 Pred, 10 Wks 35/F 19.0 Pred, 12 Wks 35/F 18.0 No 35/F 10.0 No 37/F 5.0 No>3 Mts 35/F 2.6 Pred, 3 Wks 51/F 2.0 No 31/F 0.8 Pred, 12 Wks

2.8 2.5 2.3 2.1 1.8 1.2 0.55

Treatment

Age, sex and ID50 (in 10 7 M) for Dex-inhibition of LPS-induced IL-6 production and anti-inflammatory treatment. Abbreviations: No: no antiinflammatory drugs, No>3 Mts: no anti-inflammatory drugs over the past 3 months, Wks: weeks, DSG: deoxyspergualine, Pred: methylprednisolone.

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Dickenson, Rutherford, NJ) and pooled in a 50 ml Falcon polypropylene tube. Blood was obtained between 11:00 and 16:00 h and was incubated within 2 h with LPS (Difco 055:B5, Westphal, Difco Laboratories, Detroit, MI) and/or with Dex-21-phosphate at varying concentrations (No. D1159, Sigma, St. Louis, MO) both dissolved in pyrogenfree saline (No. 314, Biofluids, Rockville, MD). To standardize the amount of blood incubated in each well in all experiments, 400 Al of blood was always added to 50 Al of LPS or saline and 50 Al of Dex or saline in a 48-well plate (No. 3548, Costar, Cambridge, MA). Final Dex concentrations in whole blood during incubation increased from zero, 10 8, 3  10 8, 10 7, 3  10 7, 10 6 to 10 5 M. After 24 h of incubation in a humidified atmosphere at 37 jC, 5% CO2, the plate was centrifuged for 10 min at 2000  g at 4 jC, the plasma was collected by pipetting and stored at 80 jC until assayed. A final dose of 30 ng/ml LPS was used in all experiments where cytokine production was inhibited with Dex. To test for cell viability after 24 h of incubation with LPS and Dex, blood was pooled after incubation, mononuclear cells isolated by Ficoll, and viability determined by Trypan Blue exclusion. No notable cell death occurred, even at the highest dose of Dex (10 5 M) as assessed after 24 h.

2.4. Lymphocyte counts Before the addition of medium to the pooled blood, a 2 ml sample was taken and total cell blood counts (CBC, 10,000 cells counted) and differential counts were made using a Cell-Dyn 3500-SL (Abbott Diagnostics, Santa Clara, CA) as routinely performed by the Haematology Laboratory of the Clinical Centre, at the NIH. 2.5. Statistics Natural log-transformed (log Dex ID50) numbers were used for statistical testing. Differences in cell counts, age, anti-inflammatory treatment or ID50 were analyzed using ANOVA, followed by Bonferroni t-tests. LPS-induced IL-6 dose – response curves were compared using repeated measurement (MANOVA). We used Pearson correlation (SAS program) to analyze the correlation between percent change in EDSS scores and log Dex ID50. Percent change in EDSS scores was calculated by subtracting EDSS score on follow up (EDSS-2) from EDSS score at enrolment (EDSS-1), divided by EDSS-1. Therefore, lower EDSS percent changes indicated lower functional status over time. In addition, the EMMIX program was used to test the existence/clustering of subgroups within MS patients.

2.3. Assays 3. Results Plasma IL-6 concentration in stimulated blood was measured using a commercial kit (CytImmune Sci., College Park, MD). This assay as previously described (Paciotti et al., 1992), with some minor modifications, is a competitive binding immunoassay based on competition between the cytokine and biotinylated cytokine for a rabbit antibody raised against the recombinant human cytokine. Briefly, 96well plates coated with a goat anti-rabbit antibody and 50 Al of plasma sample, 50 Al of assay diluent plus 25 Al of antibody against the cytokine were incubated for 3 h, followed by the addition of 25 Al of biotinylated cytokine. After an additional 30 min, the plates were washed and a conjugate of streptavidine and alkaline phosphatase was added for 30 min. The enzyme was washed out and color development achieved by adding the substrate (NADPH) 20 min later followed by an enhancer (formazan). Optical density was measured at 495 nm, while cytokine concentrations were calculated using Microplate Manager (Biorad). The detection limit was 200 pg/ml, intra-assay variability was 8 – 9%, and the inter-assay variability was 11– 12%. ID50s were determined directly from individual graphs, in which the Dex concentration leading to 50% inhibition of the maximal IL-6 production (as seen without Dex addition) was carefully determined, as described previously (DeRijk et al., 1997a). This enabled us to obtain ID50s even from patients who showed relative corticosteroid resistance. The error in determining the ID50s is within F 10%.

3.1. Dex inhibition of IL-6 production The average doses in molar and the average of the natural log Dex ID50 (Fig. 1) in each group of subjects

Fig. 1. Natural log ID50 values for Dex inhibition of LPS-induced IL-6 production in healthy controls, CPMS and RRMS patients. Blood was incubated with 30 ng/ml LPS and different doses of Dex or saline, and plasma supernatants were harvested after 24 h. An ID50 was determined from each individual dose – response curve. Log ID50s are depicted. The mean value for each group was calculated after log transformation. A significantly lower corticosteroid sensitivity was observed in the RRMS patient group compared to the controls ( P < 0.01), but not compared to the CPMS patient group. No significant difference was seen between controls and CPMS patient group. The patients, who used corticosteroids during the 3 months prior to testing, are depicted in black circles.

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Table 2 Occasion

1st

2nd

3rd

4th

40 2 30 10

21 0.6 40 40

18 3

0.5

Patient 1 2 3 4

Corticosteroid sensitivity using Dex-inhibition as measured with LPSinduced IL-6 production in four patients did not show large intra-individual differences. The numbers indicate the amount of Dex (in 10 7 M) needed to obtain 50% inhibition of LPS-induced IL-6 production.

were: healthy controls (n = 12) 3.0 F 2.0  10 7 M or 0.9 F 0.7 (mean F S.D.); CPMS group (n = 11) 6.2 F 8.8  10 7 M or 1.2 F 1.1; and in RRMS group (n = 13) 28.4 F 31.6  10 7 M or 2.6 F 1.5. The Dex ID50 in the RRMS group was significantly higher than the CPMS and control groups ( F = 7.7, P = 0.002), while no significant difference was seen between the healthy control and CPMS group. If patients who had received steroids during the 3 months prior to testing (filled circles, Fig. 1) were excluded, the difference between the RRMS group (28.3 F 27.5  10 7 M, 2.8 F 1.2, n = 8) and the CPMS (5.9 F 9.8  10 7 M, 1.1 F 1.2, n = 8) and control groups (3.0 F 2.1  10 7 M, 0.9 F 0.7, n = 12) was still significant (ANOVA: F = 9.4, P = 0.0009). Again, no significant difference was seen between the CPMS group and the control group. Importantly, anti-inflammatory drug treatment (Table 1) did not affect corticosteroid sensitivity (ANOVA: F = 0.26, P = 0.6). If viewed as a normal distribution (mean F 2 S.D.), the natural log Dex ID50 value fell outside the 95% limit in three individuals in the control group, one in the CPMS group and none in the RRMS group. In addition, no subgroups within the CPMS or RRMS groups could be detected, using the EMMIX statistical program.

Fig. 3. Blood-cell numbers in healthy controls, CPMS and RRMS patients. Blood collected for LPS stimulation and Dex-inhibition was analyzed for total white blood cell counts (WBC), granulocytes (Gran), lymphocytes (Lymph), monocytes (Mono), basophils (Baso) and Eosinophils (Eos), using an automated system (Cell-Dyn 3500-SL). Lymphocyte-numbers in CPMS patients (1232 F 118) were significantly lower than in healthy controls (1846 F 133, P<0.005) and RRMS patients (1801 F 186, P<0.05). Eosinophils were significantly lower in CPMS patients (101 F 15) compared to healthy controls (236 F 53, P<0.05) and to RRMS patients (147 F 15, P<0.05). No significant difference was observed between healthy controls and RRMS patients. Data are presented as mean F S.E.M.

These data indicate that MS patients as a group show greater variability of corticosteroid sensitivity compared to controls. Furthermore, the RRMS subgroup of patients exhibits a significantly decreased corticosteroid sensitivity compared to CPMS patients and control subjects. To determine stability of Dex-inhibited IL-6 production over time, we tested four patients on several occasions, each occasion months apart, see Table 2. The data indicate that corticosteroid sensitivity using Dex-inhibition as measured with LPS-induced IL-6 production did not show large intra-

Fig. 2. LPS induced IL-6 production in whole blood obtained from healthy controls, RRMS and CPMS patients. Blood was incubated with different doses of LPS (saline, 0.3 to 300 ng/ml), for 24 h, and plasma supernatants were collected. Significantly lower concentrations of IL-6 were found in CPMS patients compared to healthy controls ( P < 0.001) or RRMS patients ( P < 0.02). No significant difference was observed between healthy controls and RRMS patients. Data are presented as mean F S.E.M.

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Table 3 EDSS scores and Dex ID50 of subjects CPMS Age/ sex 1 2 3 4 5 6 7 8 9 10 11 12 13

RRMS ID50

54/M 30.0 29/M 15.0 42/M 4.5 39/F 4.0 55/M 3.0 41/F 2.9 39/F 2.9 46/M 2.6 41/F 1.7 47/M 1.2 43/F 0.45

EDSS-1 EDSS-2

7 6

7.5 7 5 7

7.5 8 Deceased 6.5 7.5 9 8 7.5 6.5

Age/ sex

ID50

EDSS-1 EDSS-2

41/M 100.0 3.5 49/F 79.0 31/M 60.0 2.5 33/M 30.0 1 29/F 22.0 6 42/M 21.0 35/F 19.0 1.5 35/F 18.0 1.5 35/F 10.0 0 37/F 5.0 1.5 35/F 2.6 1.5 51/F 2.0 5 31/F 0.8 2

4.5 3 6 1.5 6 2.5 1.5 2.5 2 1 1.5 3.5 3

Age and sex, ID50 (in 10 7 M) for Dex-inhibition of LPS-induced IL-6 production and EDSS-1 and -2 scores of CPMS and RRMS patients.

individual differences. If a patient had a relatively high corticosteroid sensitivity it was still high on other occasions, and if the patient showed relative corticosteroid resistance, high dosages of Dex where needed on all occasions to obtain 50% suppression. 3.2. LPS induced IL-6 production in whole blood LPS (range 0 – 300 ng/ml) induced IL-6 production showed dose-dependency and ranged from 1 ng/ml (saline incubation) up to almost 12 ng/ml (300 ng/ml LPS), Fig. 2. A small but significantly lower level of IL-6 production was

observed in the CPMS patients compared to healthy controls (repeated measurement; F = 15, P < 0.001) or compared to RRMS patients (repeated measurement; F = 5.9, P < 0.02). IL-6 production in RRMS patients was not significantly different from healthy controls. No significant differences in cell counts, in any group of cells, were observed between the healthy controls and the RRMS (Fig. 3). No consistent relationship was observed between current or prior antiinflammatory drug treatment and the capacity to produce IL6 suggesting that drug therapy could not account for the lower CPMS group IL-6 production. However, the CPMS group had significantly lower lymphocyte counts compared to healthy controls (T-test; P < 0.005) or RRMS group (T-test; P < 0.05) (Fig. 3). In addition, lower levels of eosinophils were observed in CPMS group as compared to healthy controls (T-test; P < 0.05) or RRMS group (T-test; P < 0.05) (Fig. 3). These results show that the capacity of whole blood to produce IL6 following LPS stimulation is slightly impaired in CPMS patients. 3.3. Clinical correlation Out of 24 patients, 16 patients had concomitant and follow up EDSS scores reported over approximately 6 years after original evaluation (Table 3, Fig. 4). The Pearson correlation analysis of the natural log Dex ID50 (mean of 1.6 F 1.4) and EDSS score percent change (mean of 22.6 F 42.7) revealed an r of 0.43, but the P-value at 0.1 was not statistically significant. These data indicate a trend towards an 18% negative correlation between the corticosteroid resistance and functional status over time.

Fig. 4. Scatterplot of natural log Dex ID50 and EDSS score percent change. Sixteen patients had concomitant and follow up EDSS scores reported over approximately 6 years after original evaluation. These data indicate a trend towards a negative correlation between the corticosteroid resistance and functional status over time.

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However, it should be noted that this study was not designed as a prospective long-term follow-up study. Therefore, the small number of patients who had follow-up scores most likely contributed to lack of statistical significance.

4. Discussion We found greater variability of corticosteroid sensitivity in MS patients compared to healthy controls, which was accounted for in part by the finding that RRMS as a group showed a relative corticosteroid resistance in this assay system. No significant difference in corticosteroid sensitivity was observed in CPMS patients compared to healthy controls. We also found a trend towards a negative correlation between corticosteroid resistance and functional status of the patients over time, indicating that the patients who exhibited a higher corticosteroid resistance became less functional over time. However, additional prospective studies are needed to confirm this finding. Overall, these data suggest that altered corticosteroid sensitivity may be associated with some clinical forms of MS and could be predictive of or associated with severity of symptoms. In other autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosis and asthma, changes in corticosteroid sensitivity have been described that have been postulated to contribute to the pathogenesis of these diseases (Chikanza et al., 1992; Sher et al., 1994; Tanaka et al., 1992). For example, in vitro corticosteroid sensitivity in SLE-patients was found to be positively associated with successful steroid therapy (Tanaka et al., 1992). These findings not only indicate an association between corticosteroid sensitivity and immune function, but also with clinical outcome, and suggest that degree of corticosteroid sensitivity and resistance may be an additional factor associated with susceptibility to autoimmune diseases. Corticosteroid sensitivity was measured in the study reported here, using ex vivo inhibition by Dex of LPS induced IL-6 production. LPS is a typical stimulus for monocytes and it is thought that the bulk of IL-6 is produced by cells of the macrophage-lineage in this whole blood system (Bendtzen, 1988; Akira et al., 1990). Since no differences in monocyte numbers were observed between healthy controls and MS patients, changes in corticosteroid sensitivity are probably not a result of differences in these cell numbers. However, we cannot rule out the possibility that changes in subsets of monocytes contribute to differences in corticosteroid sensitivity. In addition, no correlation between corticosteroid sensitivity and patient age, gender or therapy was observed. In previous studies, we have shown that IL-6, as measured in our ex vivo system using our specific IL-6 assay, was relatively insensitive to short-lasting changes in endogenous cortisol, such as seen during fluctuations in circadian rhythm or running stress (DeRijk et al., 1997a). In addition, repeated measures of Dex-inhibition of IL-6 (Table 2)

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indicated that corticosteroid resistance is relatively stable. This supports the finding that the high level of variability of corticosteroid sensitivity observed in the MS group may be an intrinsic property of monocytes obtained from MS patients. In addition, it is now becoming clear that corticosteroid sensitivity is much more dynamic than previously thought (Bamberger et al., 1996; DeRijk et al., 1997b; Ebrecht et al., 2000; Rohleder et al., 2003; Duclos et al., 2003). For example exercise, an acute stressor, can induce profound changes in the capacity of Dex to inhibit LPS induced IL-6 production in whole blood (DeRijk et al., 1997a; Smits et al., 1998; Duclos et al., 2003). However, in MS patients subjected to an acute psychological challenge this was not observed (Heesen et al., 2002), indicating that the mechanisms of the variability of corticosteroid sensitivity in MS remains to be elucidated. Corticosteroids are well known for their anti-inflammatory actions, especially if used at pharmacological concentrations (Cupps and Fauci, 1982). However, the effects of endogenous cortisol at physiological levels differ from their pharmacological actions (Wilckens, 1995; Wilckens and De Rijk, 1997). Recent data suggest that corticosteroids at physiological levels interfere with the time-course of T-cell proliferation, enhance T-cell proliferation (Wiegers et al., 1993, 1995), prevent apoptosis (Yang et al., 1995) and interfere with selection of T-cells (Vacchio et al., 1994; Vacchio and Ashwell, 1997). Furthermore, physiological concentrations and preparations of corticosteroids enhance delayed type hypersensitivity(Dhabhar, 2003). A dysregulation of cortisol action, brought about by changes in corticosteroid sensitivity and/or plasma cortisol, could therefore affect these regulatory actions of corticosteroids and potentially alter T-cell function in vivo and T-cell mediated autoimmune diseases at several levels. In MS patients, we previously observed an increased basal level of plasma cortisol (Michelson et al., 1994), although this study did not differentiate between CPMS and RRMS patients. In this context, the decreased IL-6 production as reported here in CPMS patients could be a result of a prolonged increase in basal levels of endogenous cortisol in the presence of a normal corticosteroid sensitivity. In addition, a normal corticosteroid sensitivity in the presence of elevated plasma cortisol could contribute to the formation of antibody producing cells in CPMS patients, since cortisol can enhance B-cell formation and antibody production (Wilckens and De Rijk, 1997). This would be consistent with the presence of antibodies in the serum of CPMS patients, which are not detected in RRMS patients (Henneberg et al., 1991, 1993). On the other hand, decreases in corticosteroid sensitivity as observed here in RRMS patients, are not associated with changes in IL-6 production, suggesting an adaptation of corticosteroid sensitivity to prolonged increases in plasma cortisol. In the absence of concurrent HPA axis response data in the patients reported here, the role of HPA axis dysregulation in the corticosteroid resistance observed cannot be determined.

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Corticosteroid sensitivity, as mediated by the glucocorticoid receptor (GR), is subject to downregulation by corticosteroids. Cytokines such as IL-1, tumor necrosis factor and the combination of IL-2 and IL-4 also decrease corticosteroid sensitivity (Kam et al., 1993; Hill et al., 1988). An alternative splicing product of the GR-gene, the GRh, has been characterized as a natural antagonist of the classic GR, GRa, which could, when present in excess, induce corticosteroid resistance (Bamberger et al., 1995). Recently, we have found that a genetic variant of the GR-gene, a single nucleotide polymorphism (SNP) in exon9h, is associated with rheumatoid arthritis (Derijk et al., 2001). We postulated that this SNP could result in corticosteroid resistance due to increased expression of the GRh-variant. Moreover, genetic studies show that primary (hereditary) abnormalities in the GR-gene are associated with relative glucocorticoid hypersensitivity in 6.6% of the normal population and relative glucocorticoid resistance in 2.3% of the population (Lamberts et al., 1996; DeRijk et al., 2002). Interestingly, the GR mediating the effects of Dex is also aberrantly regulated in EAE-susceptible LEW/N rats compared to resistant F344/N rats. Thus, adrenalectomy, known to increase GR-expression, did not increase GR-expression in LEW/N rats but did so in EAE-resistant F344/N rats (Smith et al., 1994). In addition, in MS patients the association between an HPA axis measure (plasma cortisol following the combined DexCRH-test) and GR-characteristics (the Bmax and the Kd) is no longer observed, in contrast to the strong association present in controls (Then Bergh et al., 1999). These data suggest the existence of a mismatch in MS patients between the regulation of plasma cortisol and the GR, which largely determines corticosteroid sensitivity. Thus, although the mechanisms of the changes in corticosteroid sensitivity in MS patients are currently not known, genetic, endocrine or inflammatory mechanisms could all contribute to such functional differences. Although this study was not primarily designed to be longitudinal, some follow-up clinical data over approximately 6 years after original evaluation was obtained. The Pearson correlation analysis of the natural log Dex ID50 and EDSS score percent change did not reveal a statistically significant difference ( P = 0.1), consistent with the limited number of subjects with recorded both baseline and follow up EDSS scores. The sample size required for an a of 0.05 and power of 80% is 41 subjects. Nonetheless, the data indicate a trend towards a negative correlation between the natural log Dex ID50 and EDSS score percent change. Thus, on average patients with higher corticosteroid resistance progressed to lower functional status over time. This indicates that the status of corticosteroid resistance could potentially be used to predict clinical status over time, although further studies with more subjects would be needed to confirm this finding. In conclusion, we describe a high variability of corticosteroid sensitivity in MS patients as compared to healthy controls. Moreover, if MS patients are divided into two

subgroups, RRMS patients showed a significantly lower corticosteroid sensitivity compared to controls or CPMS patients. Given the importance of endogenous cortisol in regulating immune function, these data suggest that corticosteroid sensitivity could represent a susceptibility factor in the pathogenesis and severity of MS and could potentially be used as a prognostic indicator for course and severity of disease.

Acknowledgements We thank Henry McFarland, M.D. and the members of the Neuroimmunology Branch, National Institute of Neurological Diseases and Stroke, National Institute of Health, for providing the clinical information and the specimens.

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