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The effect of season on cytokine expression in multiple sclerosis and healthy subjects
Journal of Neuroimmunology 188 (2007) 181 – 186 www.elsevier.com/locate/jneuroim
Short communication
The effect of season on cytokine expression in multiple sclerosis and healthy subjects Niall Stewart a,⁎, Bruce Taylor b , Anne-Louise Ponsonby c,d , Fotini Pittas d , Ingrid van der Mei d , Greg Woods a , Haydn Walters a a
c
School of Medicine, University of Tasmania, Locked Bag 29, Hobart 7000, Tasmania, Australia b Christchurch School of Medicine and Health Sciences, University of Otago, New Zealand National Centre for Epidemiology and Population Health, Australian National University, Australia d The Menzies Research Institute, Australia Received 17 January 2007; received in revised form 25 May 2007; accepted 5 June 2007
Abstract We measured the levels of IFNγ, TNFα, Il-4 and Il-10 produced by mitogen-stimulated peripheral blood mononuclear cells (PBMC) from healthy people, and those with the relapse/remitting form of multiple sclerosis. Blood was taken in summer and winter. Healthy people had a summer excess of Il-4, Il-10 and TNFα, and a winter excess of IFNγ. Untreated MS cases had a summer excess of Il-10, whereas those treated with Interferon-beta had lower levels of all cytokines, and displayed no seasonal effect. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; Cytokines; Season
1. Introduction Multiple sclerosis is a chronic, demyelinating inflammatory disease of the central nervous system. Its aetiology is not fully understood, but includes both genetic and environmental factors. The disease is characterised, at least in its early (inflammatory) phases by the breaking down of immunological self tolerance, with damage to the myelin sheath that may be mediated by T helper type one (Th1) cells via the secretion of the cytokines Interferon gamma (IFNγ) and Tumour Necrosis Factor alpha (TNFα) (Sharief and Hentges, 1992; Sepulcre et al., 2005). Th1 cells, and the cytokines that they produce, are thought to be important in a number of autoimmune diseases (Gysemans et al., 2005; Singh et al., 2003). In contrast, Th2 type cytokines such as Interleukin 4 (Il-4), produced by T helper 2 cells (Th2), may be beneficial, and it may be that the balance of Th1/Th2 influences is aetiologically important (Al-Shammri
⁎ Corresponding author. E-mail address: [email protected] (N. Stewart).
et al., 2004). More recently, Il-10, a generally immunosuppressive cytokine, produced by both Th2 (Al-Shammri et al., 2004) and T regulatory cells (Veldman et al., 2006) has been of particular interest (Mocellin et al., 2004). Other cytokines may also be involved in the disease process, with recent research suggesting an important role for Il-17 (Martin-Saavedra et al., 2007). Although many studies have been conducted on cytokine expression in MS and healthy subjects, no consensus has been reached on whether the cytokine profile of MS subjects intrinsically differs from that of healthy people. Age, sex, and disease type (relapse remitting, primary progressive or secondary progressive) may all affect cytokine secretion, making useful comparisons difficult (Gardner and Murasko, 2002; Nguyen et al., 2003; Eikenbloom et al., 2005). Season may also influence cytokine expression. In progressive MS, a winter peak of IFNγ and Il-12 has been observed (Balashov et al., 1998). The proinflammatory cytokine IFNγ has also been reported to increase in winter in healthy subjects (Katila et al., 1993). Here, we conduct a case control study to examine how cytokine levels vary between untreated MS cases, MS cases on
0165-5728/$ - see front matter. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2007.06.012
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N. Stewart et al. / Journal of Neuroimmunology 188 (2007) 181–186
Table 1 Numbers of subjects receiving treatment in the six months prior to blood collection Treatment
Summer
Steroids Copaxone Mitoxantrone
2 0 0
Winter 8 2 1
6 2 0
6 0 0
Subjects not receiving Interferon-beta are shown in bold, and those receiving Interferon-beta in italics. No treatments were administered in the month prior to blood collection.
discarded. Cells were then washed twice in incomplete RPMI and re-suspended in complete RPMI containing 2 mM L-glutamine (Sigma), 100U Penicillin, 100U Gentamicin (Pharmacia) and 10% FCS (JRH Biosciences) at 1 × 106 cells/ ml in 24 well cell culture plates (IWAKI). Five μg/ml PHA (Sigma) was added to each sample, and the samples were incubated for 24 h at 37C, 8% CO2. The supernatants were then collected and frozen at − 80 °C until the ELISA was performed at a single time, so as to avoid a “batching” effect. 2.3. ELISAs
immunomodulatory treatment and healthy controls. Further, we examine how cytokine levels vary by season within each group. 2. Methods 2.1. Participants Three groups of participants were recruited. Twenty six healthy controls were recruited from the Red Cross Blood Bank in Hobart Tasmania, and were age and sex matched with 25 relapsing–remitting MS patients taking part in a longitudinal study of MS progression conducted by the Menzies Research Institute. These subjects were receiving no immunomodulatory treatment. In addition, twenty four relapse–remitting subjects undergoing Interferon beta treatment were also sampled seasonally. Nineteen subjects were treated with Betaseron (250 mcg, SC alt. days), 3 with Rebif (44 mcg, SC, 3 times a week) and 2 with Avonex (30 mcg, IM, weekly). Treatment duration was for an average of 36 months. Other medications taken by the subjects are shown in Table 1. All participants were diagnosed as definite MS using the criteria described by Poser et al. (1983) and categorized using the criteria of Lublin and Reingold (1996). Blood samples for all groups were collected in summer (January, February or March) and winter (July, August or September). None of the MS subjects was suffering a relapse at the time of sampling, and none had taken steroids in the month preceding blood collection. All participants were from the Southern region of the island state of Tasmania, Latitude 43° South. Relevant environmental data for the area are shown in Table 2. This study was approved by the ethics committee of the Royal Hobart Hospital and informed consent was obtained from all participants. 2.2. Peripheral blood mononuclear cell collection and stimulation Collected blood was layered over histopaque (Sigma), spun at 400 g for 30 min, and the interface containing the PBMC was removed and washed twice in incomplete RPMI-1640 (JRH Biosciences). The cells were then resuspended in a 10% DMSO/ RPMI solution and stored at − 80 °C until required. To avoid a storage effect, as soon as a season's collection was completed, the cells were thawed and counted using a haemocytometer. Cell viability was ascertained by Trypan blue exclusion, and any samples with viability below 90% were
Samples for Il-4 and Il-10 analysis were assayed undiluted, and those for IFNγ and TNFα analyses were diluted 1:4 with Assay Diluent (BD Biosciences, San Diego). ELISAs were performed using BD OptEIA ELISA kits and reagents, as per the manufacturer's instructions. To ensure consistency, each plate (Nunc, Roskilde, Denmark) contained matched MS summer/winter samples, control samples and standards. Preliminary analyses showed that storage of supernatants for ≈ 12 months had no effect on cytokine levels. 2.4. Statistics To examine the association between variables such as sex, treatment or age on cytokine levels, multiple linear regression (Armitage and Berry, 1994) was used with adjustment for potential confounders by inclusion of covariates. For the matched case control comparison in Table 4, a general estimating equation was used (Klingenberg and Agresti, 2006; Shults et al., 2006) with clustering matched sets that compare untreated cases directly with the case's age and sex matched control. Pairwise Pearson correlation coefficients (Armitage and Berry, 1994) were used to examine the relationship between cytokine levels. A Bonferroni adjustment for multiple comparisons was made (Armitage and Berry, 1994). For the intra-individual seasonal analysis in Table 5, a general estimating equation with a logit link was used with clustering on subject identity (Klingenberg and Agresti, 2006; Shults et al., 2006). Table 2 Seasonal parameters for Hobart city (Longitude 147, Latitude − 43), Tasmania
January February March April May June July August September October November December
Mean daily temperature (°C)
Mean number of clear days
Mean daily sunshine (hours)
22 22 21 18 15 13 12 13 15 17 19 21
4.9 4.5 4.5 4.0 3.8 3.9 4.3 3.9 3.0 3.4 2.9 2.9
8.1 7.9 6.6 5.8 4.5 4.2 4.8 5.6 6.2 7.2 7.4 7.8
Australian Bureau of Meteorology (2007).
N. Stewart et al. / Journal of Neuroimmunology 188 (2007) 181–186
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3. Results 3.1. Participants Participant details are described in Table 3. 3.2. The effect of age and sex on cytokine level To determine whether cytokine levels vary by season, peripheral blood mononuclear cells from healthy subjects, untreated MS subjects and Interferon beta treated MS subjects were analysed for cytokine production in summer and winter. We first examined whether age or sex was associated with cytokine levels within each of the three groups. Among controls, age or sex was not associated with Il-4, IFNγ or TNFα levels, with or without adjustment for sex and season. Older controls had lower Il-10 levels ( p = 0.04), but this did not persist after adjustment for season and sex. Among treated or untreated MS cases, age and sex were not associated with cytokine levels for Il-4, Il-10, IFNγ or TNFα on univariate or multivariate analysis. 3.3. The cytokine profile of untreated MS cases, MS cases receiving immunomodulatory therapy and healthy controls Next we compared the cytokine levels in untreated MS cases and healthy controls, and determined the mean difference for each cytokine in summer compared to winter. There was no significant difference in cytokine levels between untreated MS cases and healthy controls, in either summer or winter. Even though no individual cytokine was associated with MS after adjusting for season, MS cases were less likely to have high Il10 relative to Il-4, and this reached borderline significance ( p = 0.07). We also examined whether the cytokine levels were lower in the treated compared to untreated MS cases after adjustment for age, sex and season. Treated cases had lower levels of Il-4, Il10, IFNγ, and TNFα (Table 4). Fig. 1. Cytokine expression by PHA-stimulated PBMC from (A) control subjects, (B) untreated MS subjects and (C) Interferon-beta treated MS subjects in summer and winter. Asterisk denotes significant difference at p b 0.05. Error bars show standard deviation.
All analyses were conducted using STATA 9.0 (Statacorp 2005: statistical software: Release 9, College Station Tex) apart from the distribution of cytokine levels examined for Fig. 1A, B and C, which were analysed by ANOVA using SPSS 13.0. Table 3 Characteristics of the study sample
n Age (mean, SD) Female (% n / N) EDSS (median, 25th, 75th percentile
Controls
MS cases (untreated)
MS cases (treated)
26 54 (9) 62 (16/26) –
25 54 (10) 64 (16/25) 5 (4,6)
24 53 (9) 67 (16/24) 4 (3,5)
3.4. Correlation between cytokines We examined correlations between the cytokines in summer and winter for healthy controls and untreated MS subjects separately (Table 5). Significant positive correlations were observed for the Th1 type cytokines IFNγ and TNFα for both Table 4 Mean differences in cytokine levels between Interferon-β treated and untreated multiple sclerosis cases Cytokine Mean differences in treated {95% Confidence Interval} p value compared to untreated MS cases (pg/ml) Il-4 Il-10 IFNγ TNFα
− 41.23 − 100.06 − 151.95 − 129.94
Adjusted for age and sex.
{− 72.38, − 10.09} {− 184.75, − 15.37} {− 200.56, − 103.35} {− 175.34, − 84.53}
0.009 0.02 b 0.0001 b 0.0001
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N. Stewart et al. / Journal of Neuroimmunology 188 (2007) 181–186
Table 5 Correlation between winter (W) and summer (S) cytokine samples for untreated multiple sclerosis cases (in bold) and controls (in italics) Il-4 (S) Il-4 (S Il-4 (W) Il-10 (S) Il-10 (W) IFNγ (S) IFNγ (W) TNFα (S) TNFα (W)
1.000 0.796 ⁎ 0.305 0.201 0.112 − 0.109 0.581 − 0.127 0.120 − 0.113 0.432 0.065 0.134 − 0.146 0.355
Il-4 (W)
Il-10 (S)
Il-10 (W)
IFNγ (S)
IFNγ (W)
TNFα (S)
TNFα (W)
1.000 − 0.058 − 0.106 − 0.054 0.340 0.078 − 0.106 0.055 0.286 0.165 0.051 0.082 0.462
1.000 0.153 0.003 0.218 0.497 0.071 − 0.288 0.120 0.281 0.105 − 0.303
1.000 0.193 0.065 0.392 0.546 0.088 0.087 0.479 0.468
1.000 0.533 − 0.079 0.610 0.783 ⁎ 0.592 − 0.155
1.000 0.434 − 0.272 0.743 ⁎ 0.772 ⁎
1.000 0.586 − 0.131
1.000
Note: Bonferonni's adjustment has been made for multiple comparisons. ⁎ pb0.001.
groups in winter, between TNFα and IFNγ for untreated MS subjects in summer and between Il-4 summer and Il-4 winter levels in healthy subjects. A significant negative correlation between the classic Th1 and Th2 cytokines, Il-4 and IFNγ, was not observed in summer or winter for either group. 3.5. Seasonal variation in cytokine levels We examined differences in cytokine levels by season. As shown in Fig. 1A, healthy subjects' levels of Il-4, Il-10 and TNFα were significantly increased in summer, and IFNγ was significantly increased in winter. Analysis of untreated MS subjects revealed similar results for Il-10 only (Fig. 1B). In Interferon-β treated subjects, there was no significant difference in mean Il-4, Il-10, IFNγ or TNFα by season (Fig. 1C). We then examined these seasonal differences by comparing winter and summer cytokine levels in an intra-individual paired analysis. Again, similar patterns were found (Table 6).
Table 6 Mean difference {95% Confidence Interval} in summer compared to winter cytokine levels in the sample groups Group
Control
Cytokine Mean difference in {95% Confidence summer compared to Interval} winter levels (pg/ml)
Il-4 Il-10 IFNγ TNFα Untreated MS Il-4 Il-10 IFNγ TNFα Treated MS Il-4 Il-10 IFNγ TNFα
25.28 116.25 − 54.03 68.18 38.97 138.55 3.61 − 10.00 0.81 101.98 1.92 42.71
{3.64, 46.93} {22.73, 209.77} {− 106.22, − 1.94} {24.83, 111.54} {− 4.28, 52.22} {19.09, 258.01} {− 61.46, 68.67} {− 74.28, 54.28} {− 30.71, − 32.34} {− 3.25, 207.23} {− 30.76, 34.60} {− 20.95, 106.36}
p value
0.02 0.02 0.04 0.002 0.08 0.02 0.91 0.76 0.96 0.06 0.91 0.19
Mean summer/winter differences did not vary significantly across the three groups.
4. Discussion In this study, the cytokine profile of untreated MS cases and controls was similar, but among MS cases, treated cases had lower cytokine levels for Il-4, Il-10, IFNγ and TNFα. Marked seasonal differences in cytokine levels were observed with a summer excess of Il-10 among untreated MS cases and controls, and a winter excess of IFNγ among healthy controls, as previously reported (Katila et al., 1993; Shirai et al., 2003). This study supports the hypothesis that there is a shift in cytokine profile from winter to summer. The summer increase in Il-10 is consistent with past work showing that serum 25OHD levels, largely UVR-derived, from cord blood correlate positively with Il-10 levels (Zittermann et al., 2004) and children born in summer compared with winter have a significantly higher Il-10 to total IgE ratio (Zittermann et al., 2004). To our knowledge this is the first time an Il-10 increase in summer has been documented for untreated MS cases. The Th1 cytokine TNFα did not vary for untreated MS cases, whereas it peaked in summer in healthy subjects. The role of this cytokine in MS is still controversial, with some evidence that it is pro-inflammatory in early stages of the disease, but later exerts anti-inflammatory properties (Kassiotis and Kollias, 2001). Significant case control differences in cytokine levels were not found. As has previously been reported, IFNγ and TNFα levels increase during exacerbations (Hollifield et al., 2003; Correale et al., 2006), as would be expected in an active immune response, but our data suggest that during remission levels return to normal (equivalent to healthy subject) levels. We found no inverse association between Th1 and Th2 cytokine levels. For example, the “classic” Th1 and Th2 cytokines, IFNγ and Il-4, were not significantly negatively correlated in either untreated MS cases or healthy controls. This suggests that the association between Th1 and Th2 activity is more complex than one involving only negative feedback. The use of Interferon-beta removed any seasonal effect on cytokine expression, and suppressed both pro- and anti-
N. Stewart et al. / Journal of Neuroimmunology 188 (2007) 181–186
inflammatory cytokines, although the suppression was greater on IFNγ and TNFα. Other studies have also found that IFN-β suppresses IFNγ and TNFα (Mei et al., 2006; Ossege et al., 2001). However, the data on Il-10 are inconsistent. In in vitro systems in which IFN-β was added to PBMC cultures, Il-10 levels were increased (Huang et al., 2001). Similarly, PBMC from MS subjects treated with IFN-β, when stimulated with Staphylococcus aureus, produced more Il-10 (Byrnes et al., 2002). However, Waubant et al. (2001) report lower serum Il-10 levels in IFN-β treated subjects. Many of the difference may be explained by different methodologies, but what is clear is that IFN-β suppresses the inflammatory cytokines IFNγ and TNFα more than the anti-inflammatory Il-4 and Il-10. None of the subjects was tested for neutralizing antibodies to IFN-β, which may have affected the results (Bertolotto et al., 2002). Thus, the results should be interpreted as unadjusted for additional cytokine alterations due not to the treatment, but to associated neutralizing antibodies. How the seasonal effect noted in this study is mediated is not known. It is possible that Vitamin D plays an important role. This secosteroid is a powerful immunosuppressant and bodily stores are higher in summer than winter, due to changes in the levels of ultraviolet radiation throughout the year. Thus seasonal levels of Vitamin D may affect seasonal levels of cytokines. We are currently analysing the possible interactions between serum Vitamin D levels and Interferon-beta treatment. Although the study was conducted on frozen, not fresh PBMC, we do not think this influenced the study findings. Cryopreservation of cells allowed all assays to be conducted concurrently, thus avoiding inter-assay variation (Kvarnstrom et al., 2004). The reports of the effects of cryopreservation on cytokine expression are contradictory, but it has been reported that it does not affect the proportion of CD4+ T cells, CD8+ T cells, CD14+ monocytes and CD16+ NK cells (Allsopp et al., 1998). Moreover, Il-10 and IFNγ secretion has been shown to be unaffected by cryopreservation in samples from MS subjects (Kvarnstrom et al., 2004). Whilst conceding that cryopreservation may affect cytokine expression, as all the cells were treated identically and each plate contained all the comparison groups of interest, the differences seen by season, case or treatment are unlikely to have been influenced by the process. In conclusion, seasonal differences in cytokine patterns were observed for untreated MS cases and controls in Tasmania, Australia. The higher Il-10 levels in summer compared to winter are of interest given that Il-10 is generally an immunosuppressive cytokine and further work is required to relate this to other factors that are more common in summer, such as ultraviolet radiation exposure. References Allsopp, C.E., Nicholls, S.J., Langhorne, J., 1998. A flow cytometric method to assess antigen-specific proliferative responses of different subpopulations of fresh and cryopreserved human peripheral blood mononuclear cells. J. Immunol. Methods 214, 175–186. Al-Shammri, S, Rawoot, P, Azizieh, F, AbuQoora, A, Hanna, M, Saminathan, et al., 2004. Th1/Th2 cytokine patterns and clinical profiles during and after pregnancy in women with multiple sclerosis. J. Neurol. Sci. 222, 21–27.
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Short communication
The effect of season on cytokine expression in multiple sclerosis and healthy subjects Niall Stewart a,⁎, Bruce Taylor b , Anne-Louise Ponsonby c,d , Fotini Pittas d , Ingrid van der Mei d , Greg Woods a , Haydn Walters a a
c
School of Medicine, University of Tasmania, Locked Bag 29, Hobart 7000, Tasmania, Australia b Christchurch School of Medicine and Health Sciences, University of Otago, New Zealand National Centre for Epidemiology and Population Health, Australian National University, Australia d The Menzies Research Institute, Australia Received 17 January 2007; received in revised form 25 May 2007; accepted 5 June 2007
Abstract We measured the levels of IFNγ, TNFα, Il-4 and Il-10 produced by mitogen-stimulated peripheral blood mononuclear cells (PBMC) from healthy people, and those with the relapse/remitting form of multiple sclerosis. Blood was taken in summer and winter. Healthy people had a summer excess of Il-4, Il-10 and TNFα, and a winter excess of IFNγ. Untreated MS cases had a summer excess of Il-10, whereas those treated with Interferon-beta had lower levels of all cytokines, and displayed no seasonal effect. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. Keywords: Multiple sclerosis; Cytokines; Season
1. Introduction Multiple sclerosis is a chronic, demyelinating inflammatory disease of the central nervous system. Its aetiology is not fully understood, but includes both genetic and environmental factors. The disease is characterised, at least in its early (inflammatory) phases by the breaking down of immunological self tolerance, with damage to the myelin sheath that may be mediated by T helper type one (Th1) cells via the secretion of the cytokines Interferon gamma (IFNγ) and Tumour Necrosis Factor alpha (TNFα) (Sharief and Hentges, 1992; Sepulcre et al., 2005). Th1 cells, and the cytokines that they produce, are thought to be important in a number of autoimmune diseases (Gysemans et al., 2005; Singh et al., 2003). In contrast, Th2 type cytokines such as Interleukin 4 (Il-4), produced by T helper 2 cells (Th2), may be beneficial, and it may be that the balance of Th1/Th2 influences is aetiologically important (Al-Shammri
⁎ Corresponding author. E-mail address: [email protected] (N. Stewart).
et al., 2004). More recently, Il-10, a generally immunosuppressive cytokine, produced by both Th2 (Al-Shammri et al., 2004) and T regulatory cells (Veldman et al., 2006) has been of particular interest (Mocellin et al., 2004). Other cytokines may also be involved in the disease process, with recent research suggesting an important role for Il-17 (Martin-Saavedra et al., 2007). Although many studies have been conducted on cytokine expression in MS and healthy subjects, no consensus has been reached on whether the cytokine profile of MS subjects intrinsically differs from that of healthy people. Age, sex, and disease type (relapse remitting, primary progressive or secondary progressive) may all affect cytokine secretion, making useful comparisons difficult (Gardner and Murasko, 2002; Nguyen et al., 2003; Eikenbloom et al., 2005). Season may also influence cytokine expression. In progressive MS, a winter peak of IFNγ and Il-12 has been observed (Balashov et al., 1998). The proinflammatory cytokine IFNγ has also been reported to increase in winter in healthy subjects (Katila et al., 1993). Here, we conduct a case control study to examine how cytokine levels vary between untreated MS cases, MS cases on
0165-5728/$ - see front matter. Crown Copyright © 2007 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.jneuroim.2007.06.012
182
N. Stewart et al. / Journal of Neuroimmunology 188 (2007) 181–186
Table 1 Numbers of subjects receiving treatment in the six months prior to blood collection Treatment
Summer
Steroids Copaxone Mitoxantrone
2 0 0
Winter 8 2 1
6 2 0
6 0 0
Subjects not receiving Interferon-beta are shown in bold, and those receiving Interferon-beta in italics. No treatments were administered in the month prior to blood collection.
discarded. Cells were then washed twice in incomplete RPMI and re-suspended in complete RPMI containing 2 mM L-glutamine (Sigma), 100U Penicillin, 100U Gentamicin (Pharmacia) and 10% FCS (JRH Biosciences) at 1 × 106 cells/ ml in 24 well cell culture plates (IWAKI). Five μg/ml PHA (Sigma) was added to each sample, and the samples were incubated for 24 h at 37C, 8% CO2. The supernatants were then collected and frozen at − 80 °C until the ELISA was performed at a single time, so as to avoid a “batching” effect. 2.3. ELISAs
immunomodulatory treatment and healthy controls. Further, we examine how cytokine levels vary by season within each group. 2. Methods 2.1. Participants Three groups of participants were recruited. Twenty six healthy controls were recruited from the Red Cross Blood Bank in Hobart Tasmania, and were age and sex matched with 25 relapsing–remitting MS patients taking part in a longitudinal study of MS progression conducted by the Menzies Research Institute. These subjects were receiving no immunomodulatory treatment. In addition, twenty four relapse–remitting subjects undergoing Interferon beta treatment were also sampled seasonally. Nineteen subjects were treated with Betaseron (250 mcg, SC alt. days), 3 with Rebif (44 mcg, SC, 3 times a week) and 2 with Avonex (30 mcg, IM, weekly). Treatment duration was for an average of 36 months. Other medications taken by the subjects are shown in Table 1. All participants were diagnosed as definite MS using the criteria described by Poser et al. (1983) and categorized using the criteria of Lublin and Reingold (1996). Blood samples for all groups were collected in summer (January, February or March) and winter (July, August or September). None of the MS subjects was suffering a relapse at the time of sampling, and none had taken steroids in the month preceding blood collection. All participants were from the Southern region of the island state of Tasmania, Latitude 43° South. Relevant environmental data for the area are shown in Table 2. This study was approved by the ethics committee of the Royal Hobart Hospital and informed consent was obtained from all participants. 2.2. Peripheral blood mononuclear cell collection and stimulation Collected blood was layered over histopaque (Sigma), spun at 400 g for 30 min, and the interface containing the PBMC was removed and washed twice in incomplete RPMI-1640 (JRH Biosciences). The cells were then resuspended in a 10% DMSO/ RPMI solution and stored at − 80 °C until required. To avoid a storage effect, as soon as a season's collection was completed, the cells were thawed and counted using a haemocytometer. Cell viability was ascertained by Trypan blue exclusion, and any samples with viability below 90% were
Samples for Il-4 and Il-10 analysis were assayed undiluted, and those for IFNγ and TNFα analyses were diluted 1:4 with Assay Diluent (BD Biosciences, San Diego). ELISAs were performed using BD OptEIA ELISA kits and reagents, as per the manufacturer's instructions. To ensure consistency, each plate (Nunc, Roskilde, Denmark) contained matched MS summer/winter samples, control samples and standards. Preliminary analyses showed that storage of supernatants for ≈ 12 months had no effect on cytokine levels. 2.4. Statistics To examine the association between variables such as sex, treatment or age on cytokine levels, multiple linear regression (Armitage and Berry, 1994) was used with adjustment for potential confounders by inclusion of covariates. For the matched case control comparison in Table 4, a general estimating equation was used (Klingenberg and Agresti, 2006; Shults et al., 2006) with clustering matched sets that compare untreated cases directly with the case's age and sex matched control. Pairwise Pearson correlation coefficients (Armitage and Berry, 1994) were used to examine the relationship between cytokine levels. A Bonferroni adjustment for multiple comparisons was made (Armitage and Berry, 1994). For the intra-individual seasonal analysis in Table 5, a general estimating equation with a logit link was used with clustering on subject identity (Klingenberg and Agresti, 2006; Shults et al., 2006). Table 2 Seasonal parameters for Hobart city (Longitude 147, Latitude − 43), Tasmania
January February March April May June July August September October November December
Mean daily temperature (°C)
Mean number of clear days
Mean daily sunshine (hours)
22 22 21 18 15 13 12 13 15 17 19 21
4.9 4.5 4.5 4.0 3.8 3.9 4.3 3.9 3.0 3.4 2.9 2.9
8.1 7.9 6.6 5.8 4.5 4.2 4.8 5.6 6.2 7.2 7.4 7.8
Australian Bureau of Meteorology (2007).
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3. Results 3.1. Participants Participant details are described in Table 3. 3.2. The effect of age and sex on cytokine level To determine whether cytokine levels vary by season, peripheral blood mononuclear cells from healthy subjects, untreated MS subjects and Interferon beta treated MS subjects were analysed for cytokine production in summer and winter. We first examined whether age or sex was associated with cytokine levels within each of the three groups. Among controls, age or sex was not associated with Il-4, IFNγ or TNFα levels, with or without adjustment for sex and season. Older controls had lower Il-10 levels ( p = 0.04), but this did not persist after adjustment for season and sex. Among treated or untreated MS cases, age and sex were not associated with cytokine levels for Il-4, Il-10, IFNγ or TNFα on univariate or multivariate analysis. 3.3. The cytokine profile of untreated MS cases, MS cases receiving immunomodulatory therapy and healthy controls Next we compared the cytokine levels in untreated MS cases and healthy controls, and determined the mean difference for each cytokine in summer compared to winter. There was no significant difference in cytokine levels between untreated MS cases and healthy controls, in either summer or winter. Even though no individual cytokine was associated with MS after adjusting for season, MS cases were less likely to have high Il10 relative to Il-4, and this reached borderline significance ( p = 0.07). We also examined whether the cytokine levels were lower in the treated compared to untreated MS cases after adjustment for age, sex and season. Treated cases had lower levels of Il-4, Il10, IFNγ, and TNFα (Table 4). Fig. 1. Cytokine expression by PHA-stimulated PBMC from (A) control subjects, (B) untreated MS subjects and (C) Interferon-beta treated MS subjects in summer and winter. Asterisk denotes significant difference at p b 0.05. Error bars show standard deviation.
All analyses were conducted using STATA 9.0 (Statacorp 2005: statistical software: Release 9, College Station Tex) apart from the distribution of cytokine levels examined for Fig. 1A, B and C, which were analysed by ANOVA using SPSS 13.0. Table 3 Characteristics of the study sample
n Age (mean, SD) Female (% n / N) EDSS (median, 25th, 75th percentile
Controls
MS cases (untreated)
MS cases (treated)
26 54 (9) 62 (16/26) –
25 54 (10) 64 (16/25) 5 (4,6)
24 53 (9) 67 (16/24) 4 (3,5)
3.4. Correlation between cytokines We examined correlations between the cytokines in summer and winter for healthy controls and untreated MS subjects separately (Table 5). Significant positive correlations were observed for the Th1 type cytokines IFNγ and TNFα for both Table 4 Mean differences in cytokine levels between Interferon-β treated and untreated multiple sclerosis cases Cytokine Mean differences in treated {95% Confidence Interval} p value compared to untreated MS cases (pg/ml) Il-4 Il-10 IFNγ TNFα
− 41.23 − 100.06 − 151.95 − 129.94
Adjusted for age and sex.
{− 72.38, − 10.09} {− 184.75, − 15.37} {− 200.56, − 103.35} {− 175.34, − 84.53}
0.009 0.02 b 0.0001 b 0.0001
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Table 5 Correlation between winter (W) and summer (S) cytokine samples for untreated multiple sclerosis cases (in bold) and controls (in italics) Il-4 (S) Il-4 (S Il-4 (W) Il-10 (S) Il-10 (W) IFNγ (S) IFNγ (W) TNFα (S) TNFα (W)
1.000 0.796 ⁎ 0.305 0.201 0.112 − 0.109 0.581 − 0.127 0.120 − 0.113 0.432 0.065 0.134 − 0.146 0.355
Il-4 (W)
Il-10 (S)
Il-10 (W)
IFNγ (S)
IFNγ (W)
TNFα (S)
TNFα (W)
1.000 − 0.058 − 0.106 − 0.054 0.340 0.078 − 0.106 0.055 0.286 0.165 0.051 0.082 0.462
1.000 0.153 0.003 0.218 0.497 0.071 − 0.288 0.120 0.281 0.105 − 0.303
1.000 0.193 0.065 0.392 0.546 0.088 0.087 0.479 0.468
1.000 0.533 − 0.079 0.610 0.783 ⁎ 0.592 − 0.155
1.000 0.434 − 0.272 0.743 ⁎ 0.772 ⁎
1.000 0.586 − 0.131
1.000
Note: Bonferonni's adjustment has been made for multiple comparisons. ⁎ pb0.001.
groups in winter, between TNFα and IFNγ for untreated MS subjects in summer and between Il-4 summer and Il-4 winter levels in healthy subjects. A significant negative correlation between the classic Th1 and Th2 cytokines, Il-4 and IFNγ, was not observed in summer or winter for either group. 3.5. Seasonal variation in cytokine levels We examined differences in cytokine levels by season. As shown in Fig. 1A, healthy subjects' levels of Il-4, Il-10 and TNFα were significantly increased in summer, and IFNγ was significantly increased in winter. Analysis of untreated MS subjects revealed similar results for Il-10 only (Fig. 1B). In Interferon-β treated subjects, there was no significant difference in mean Il-4, Il-10, IFNγ or TNFα by season (Fig. 1C). We then examined these seasonal differences by comparing winter and summer cytokine levels in an intra-individual paired analysis. Again, similar patterns were found (Table 6).
Table 6 Mean difference {95% Confidence Interval} in summer compared to winter cytokine levels in the sample groups Group
Control
Cytokine Mean difference in {95% Confidence summer compared to Interval} winter levels (pg/ml)
Il-4 Il-10 IFNγ TNFα Untreated MS Il-4 Il-10 IFNγ TNFα Treated MS Il-4 Il-10 IFNγ TNFα
25.28 116.25 − 54.03 68.18 38.97 138.55 3.61 − 10.00 0.81 101.98 1.92 42.71
{3.64, 46.93} {22.73, 209.77} {− 106.22, − 1.94} {24.83, 111.54} {− 4.28, 52.22} {19.09, 258.01} {− 61.46, 68.67} {− 74.28, 54.28} {− 30.71, − 32.34} {− 3.25, 207.23} {− 30.76, 34.60} {− 20.95, 106.36}
p value
0.02 0.02 0.04 0.002 0.08 0.02 0.91 0.76 0.96 0.06 0.91 0.19
Mean summer/winter differences did not vary significantly across the three groups.
4. Discussion In this study, the cytokine profile of untreated MS cases and controls was similar, but among MS cases, treated cases had lower cytokine levels for Il-4, Il-10, IFNγ and TNFα. Marked seasonal differences in cytokine levels were observed with a summer excess of Il-10 among untreated MS cases and controls, and a winter excess of IFNγ among healthy controls, as previously reported (Katila et al., 1993; Shirai et al., 2003). This study supports the hypothesis that there is a shift in cytokine profile from winter to summer. The summer increase in Il-10 is consistent with past work showing that serum 25OHD levels, largely UVR-derived, from cord blood correlate positively with Il-10 levels (Zittermann et al., 2004) and children born in summer compared with winter have a significantly higher Il-10 to total IgE ratio (Zittermann et al., 2004). To our knowledge this is the first time an Il-10 increase in summer has been documented for untreated MS cases. The Th1 cytokine TNFα did not vary for untreated MS cases, whereas it peaked in summer in healthy subjects. The role of this cytokine in MS is still controversial, with some evidence that it is pro-inflammatory in early stages of the disease, but later exerts anti-inflammatory properties (Kassiotis and Kollias, 2001). Significant case control differences in cytokine levels were not found. As has previously been reported, IFNγ and TNFα levels increase during exacerbations (Hollifield et al., 2003; Correale et al., 2006), as would be expected in an active immune response, but our data suggest that during remission levels return to normal (equivalent to healthy subject) levels. We found no inverse association between Th1 and Th2 cytokine levels. For example, the “classic” Th1 and Th2 cytokines, IFNγ and Il-4, were not significantly negatively correlated in either untreated MS cases or healthy controls. This suggests that the association between Th1 and Th2 activity is more complex than one involving only negative feedback. The use of Interferon-beta removed any seasonal effect on cytokine expression, and suppressed both pro- and anti-
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inflammatory cytokines, although the suppression was greater on IFNγ and TNFα. Other studies have also found that IFN-β suppresses IFNγ and TNFα (Mei et al., 2006; Ossege et al., 2001). However, the data on Il-10 are inconsistent. In in vitro systems in which IFN-β was added to PBMC cultures, Il-10 levels were increased (Huang et al., 2001). Similarly, PBMC from MS subjects treated with IFN-β, when stimulated with Staphylococcus aureus, produced more Il-10 (Byrnes et al., 2002). However, Waubant et al. (2001) report lower serum Il-10 levels in IFN-β treated subjects. Many of the difference may be explained by different methodologies, but what is clear is that IFN-β suppresses the inflammatory cytokines IFNγ and TNFα more than the anti-inflammatory Il-4 and Il-10. None of the subjects was tested for neutralizing antibodies to IFN-β, which may have affected the results (Bertolotto et al., 2002). Thus, the results should be interpreted as unadjusted for additional cytokine alterations due not to the treatment, but to associated neutralizing antibodies. How the seasonal effect noted in this study is mediated is not known. It is possible that Vitamin D plays an important role. This secosteroid is a powerful immunosuppressant and bodily stores are higher in summer than winter, due to changes in the levels of ultraviolet radiation throughout the year. Thus seasonal levels of Vitamin D may affect seasonal levels of cytokines. We are currently analysing the possible interactions between serum Vitamin D levels and Interferon-beta treatment. Although the study was conducted on frozen, not fresh PBMC, we do not think this influenced the study findings. Cryopreservation of cells allowed all assays to be conducted concurrently, thus avoiding inter-assay variation (Kvarnstrom et al., 2004). The reports of the effects of cryopreservation on cytokine expression are contradictory, but it has been reported that it does not affect the proportion of CD4+ T cells, CD8+ T cells, CD14+ monocytes and CD16+ NK cells (Allsopp et al., 1998). Moreover, Il-10 and IFNγ secretion has been shown to be unaffected by cryopreservation in samples from MS subjects (Kvarnstrom et al., 2004). Whilst conceding that cryopreservation may affect cytokine expression, as all the cells were treated identically and each plate contained all the comparison groups of interest, the differences seen by season, case or treatment are unlikely to have been influenced by the process. In conclusion, seasonal differences in cytokine patterns were observed for untreated MS cases and controls in Tasmania, Australia. The higher Il-10 levels in summer compared to winter are of interest given that Il-10 is generally an immunosuppressive cytokine and further work is required to relate this to other factors that are more common in summer, such as ultraviolet radiation exposure. References Allsopp, C.E., Nicholls, S.J., Langhorne, J., 1998. A flow cytometric method to assess antigen-specific proliferative responses of different subpopulations of fresh and cryopreserved human peripheral blood mononuclear cells. J. Immunol. Methods 214, 175–186. Al-Shammri, S, Rawoot, P, Azizieh, F, AbuQoora, A, Hanna, M, Saminathan, et al., 2004. Th1/Th2 cytokine patterns and clinical profiles during and after pregnancy in women with multiple sclerosis. J. Neurol. Sci. 222, 21–27.
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