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IL-15 is elevated in serum and cerebrospinal fluid of patients with multiple sclerosis
Journal of the Neurological Sciences 241 (2006) 25 – 29 www.elsevier.com/locate/jns
IL-15 is elevated in serum and cerebrospinal fluid of patients with multiple sclerosis M. Rentzos a,*, C. Cambouri b, A. Rombos a, C. Nikolaou b, M. Anagnostouli a, A. Tsoutsou b, A. Dimitrakopoulos a, N. Triantafyllou a, D. Vassilopoulos a a
Department of Neurology, Aeginition Hospital-Athens Medical School, 72-74 Vas.So phias Av, Greece b Department of Biopathology-Immunology, Aeginition Hospital-Athens Medical School, Greece Received 11 May 2005; received in revised form 26 August 2005; accepted 5 October 2005 Available online 28 November 2005
Abstract Interleukin-15 (IL-15) is a novel proinflammatory cytokine having similar biological activities to IL-2 which is implicated in the pathogenesis of multiple sclerosis. It is produced by activated blood monocytes, macrophages and glial cells. There is little information about the involvement of IL-15 in the development of multiple sclerosis (MS). The objective of our study was to measure IL-15 serum and cerebrospinal fluid (CSF) levels in MS patients and to correlate serum and CSF IL-15 concentrations with clinical parameters of the disease. CSF IL-15/Serum IL-15 ratio (c/s IL-15 ratio) was introduced to assess the origin of elevated IL-15 levels. Materials and methods: We measured serum and CSF IL-15 levels in 52 patients with MS and 36 age and gender matched patients with inflammatory (IND) and non-inflammatory neurological diseases (NIND) studied as control groups. IL-15 levels were correlated with clinical parameters as duration, disability, MRI activity and clinical subtypes of the disease. Results: MS patients were found to have significantly higher serum IL-15 levels compared with IND ( p = 0.00016) and NIND patients ( p = 0.00045). Elevated levels of IL-15 were also found in CSF samples from MS patients compared with patients with IND ( p = 0.00034) and NIND ( p = 0.0003). Among MS subgroups there were no statistically different IL-15 serum and CSF concentrations. No significant correlation of serum and CSF IL-15 concentrations with MRI activity, disability assessed by EDSS score and duration of the disease were also found. C/S IL-15 ratio was found lower in MS patients compared with IND ( p = 0.01) and not significantly different compared with NIND patients ( p = 0.14) suggesting that systemic activation might be the source of high CSF IL-15 levels in MS patients. Conclusions: Our findings suggest a possible role of IL-15 in the immunopathogenetic mechanisms of MS. D 2005 Elsevier B.V. All rights reserved. Keywords: IL-15; Multiple sclerosis; Clinical parameters; Immunopathogenetic mechanisms
1. Introduction Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS). Its cause remains unknown, although there is evidence of a complex and multifactorial aetiology with an underlying genetic susceptibility associated with unknown environmental factors [1]. Lymphocyte activation, extravasation and infiltration into the CNS together with activation of potentially immunocompetent
* Corresponding author. Tel.: +30 210 7289200; fax: +30 210 7219860. E-mail address: [email protected] (M. Rentzos). 0022-510X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2005.10.003
glial cells are considered to lead to myelin destruction [2,3]. Proinflammatory T-Helper (TH)-1 cytokines such as Tumor Necrosis Factor, Interferon-g and Interleukin-2 seem to be related to inflammation and tissue damage in MS [4 – 7]. In the contrary, TH2/TH3 cytokines may contribute to suppress disease activity and progression [8 –10]. IL-15 is a proinflammatory cytokine. Its three-dimensional structure is similar to that of IL-2, although they do not share a significant homology. Their receptors share h and g chains, but IL-15 receptor a-chain is unique [11]. IL15 is produced by activated blood monocytes, macrophages, dendritic cells and activated glial cells [12 – 16]. It promotes T cell proliferation, induction of cytolytic effector cells
26
M. Rentzos et al. / Journal of the Neurological Sciences 241 (2006) 25 – 29
including natural killer (NK) and cytotoxic cells and stimulates B-cell to proliferate and secrete immunoglobulins [6,17]. Little information is available on the exact role of IL-15 in the development of MS lesions [10,18 –20]. Our study is focused on IL-15 production in MS patients, as well as on possible correlations between serum and cerebrospinal fluid (CSF) IL-15 levels and clinical subtype of the disease, presence of active lesions in MRI, duration of the disease and disability status.
2. Materials and methods 2.1. Patients Serum samples were studied in 80 individuals comprising the following groups: (1) 52 patients with MS (25 men and 27 women, aged 18 to 68 years, mean 35); (2) 20 agematched patients with other non-inflammatory neurological diseases (NIND); 5 out of these had amyotrophic lateral sclerosis (ALS), 3 had dementia, 3 had Parkinson’s disease and 10 had epilepsy; (3) 16 age-matched patients with other inflammatory neurological diseases (IND); 12 out of these had Guillain –Barre` Syndrome (GBS) and 4 had Chronic Inflammatory Demyelinating Polyneuropathy (CIDP). The second and third groups were studied as a control group. All of MS patients had definite MS according to the criteria of McDonald et al. [21] except for 6 patients that had isolated demyelinating syndrome (IDS) and were at high risk for the development of multiple sclerosis. In IDS group, patients with optic neuritis, diplopia, hemiparesis and cerebellar ataxia were included. These patients presented the first demyelinating event and had two or more clinically silent brain lesions on MRI [22]. MS patients were scored by the Expanded Disability Status Scale (EDSS) [23]. Mean EDSS score was 3.0 T 1.4, range 1.5 – 7.5. They were divided into two groups: 32 patients had EDSS < 3.5 and 14 had EDSS > 3.5. The mean duration of the disease was 7 T 7.5, range 0.08– 43 years. 30 patients had duration of the disease <10 years and 16 > 10 years. Of the 46 patients with MS 29 had the relapsing –remitting (RR) type of the disease, 13 had the secondary progressive (SP), and 4 the primary progressive (PP) type of the disease. MS patients were also divided into two other groups: 9 with brain or spinal cord magnetic resonance imaging (MRI) activity and 37 without. MS patients were considered to have MRI activity when they had one or more enhancing lesions in T1-weighted spin-echo images after gadopentate dimeglumine (Gd-DTPA) injection. GdDTPA was given intravenously at a dose of 0.1 mmol/kg and about 15 min after contrast injection a T1-weighted sequence was repeated. Ethical consent has been obtained by all patients and controls for studying IL-15 serum and CSF levels. Serum samples were stored at 70 -C until assayed. Cerebrospinal fluid (CSF) samples of patients with
MS, NIND and IND were obtained by lumbar puncture. CSF was centrifuged to separate the cells and then frozen within 2 h in multiple aliquots at 70 -C. No patient with MS had received any immunomodulatory treatment for at least 3 months prior to study. 2.2. Serum and CSF IL-15 assays Serum and CSF concentrations of circulating IL-15 were measured by immunoassay using commercially available enzyme-like immunosorbent assay (ELISA) kits (R&D systems). Undiluted serum and CSF samples were added in duplicate to microtiter wells and assayed according to routine procedures. Optical densities were determined by means of a micro-ELISA reader (L.P 400, Pasteur diagnostics). Values were calculated by comparison with a standard curve that was generated with IL-15 standards. The limit of detection was 2 pg/ml.
3. Statistical analysis Results were expressed as mean T standard deviation. The Student’s t-test and the Mann – Whitney U-test were used, as appropriate, to compare titres of IL-15 between the examined study groups. Data are expressed as the mean T S.D. All comparisons were two-sided, with a pvalue of less than 0.05 used to indicate statistical significance. The statistical software used for this analysis was Statistica 6.0.
4. Results 4.1. Serum IL-15 levels Serum IL-15 levels were significantly elevated in the MS group (mean T standard deviation [S.D.], 4.2 T 1.36 pg/ml) compared with patients with IND (1.95 T 0.41 pg/ml; p = 0.00016) and NIND (2.96 T 1.13 pg/ml; p = 0.00045 (Fig. 1). Serum IL-15 levels were not significantly different in RRMS patients (4.13 T 0.61 pg/ml) compared with SPSM patients (4.26 T 0.73 pg/ml; p = 0.64) and IDS (4.13 T 0.45 pg/ml; p = 0.99) (Fig. 2). PPMS patients (5.56 T 3.6 pg/ml) had higher IL-15 values compared to patients with all other types of the disease (4.15 T 0.6 pg/ml; p = 0.018) (Fig. 2). Among MS patients, patients with MRI activity (mean T S.D., 3.97 T 0.57 pg/ml) had no significantly different serum IL-15 levels when compared with patients with no MRI activity (4.38 T 1.54 pg/ml, p = ns). IL-15 levels were not also different in patients with over 10 years duration of the disease (4.92 T 0.56 pg/ml) compared with those under 10 years (4.1 T 0.62 pg/ml, p = ns) or patients with more disabling disease (EDSS > 3.5) (5.00 T 2.64 pg/ml) compared with those with mild disability (EDSS < 3.5) (4.1 T 0.81 pg/ ml, p = ns).
M. Rentzos et al. / Journal of the Neurological Sciences 241 (2006) 25 – 29 6,0 5,5
Mean
+SD
5,0
c IL-15
s IL-15
4,5 4,0 3,5 3,0 2,5 2,0 1,5 1,0
MS
IND
NIND
Fig. 1. Distribution of IL-15 levels in serum samples of patients with MS, IND and NIND. MS = multiple sclerosis, IND = inflammatory neurological diseases, NIND = non-inflammatory neurological diseases.
4.2. CSF IL-15 levels Elevated levels of IL-15 were found in CSF samples from patients with MS (mean T S.D., 3.27 T 0.91 pg/ml) compared with IND (2 T 0.47 pg/ml; p = 0.00034) and NIND patients (2.43 T 0.49 pg/ml; p = 0.00029) (Fig. 3). CSF IL-15 levels were not significantly different in RRMS patients (3.34 T 0.9 pg/ml) compared with SPMS (3.31 T1.03 pg/ml; p = 0.93), IDS (3.5 T 0.84 pg/ml; p = 0.7) and PPMS patients (3.43 T 0.8 pg/ml; p = 0.83). Among MS patients, patients with MRI activity (mean T S.D., 3.12 T 1.22 pg/ml) had no significantly different CSF IL-15 levels when compared with patients with no MRI activity (3.38 T 0.88 pg/ml, p = ns). IL-15 levels were not also different in patients with over 10 years duration of the disease (3.6 T 0.71 pg/ml) compared with those under 10 years (3.21 T 0.48 pg/ml, p = ns) or patients with more
4,4 4,2 4,0 3,8 3,6 3,4 3,2 3,0 2,8 2,6 2,4 2,2 2,0 1,8 1,6 1,4
27
Mean
MS
IND
+SD
NIND
Fig. 3. Distribution of IL-15 levels in CSF samples of patients with MS, IND and NIND. MS = multiple sclerosis, IND = inflammatory neurological diseases, NIND = non-inflammatory neurological diseases.
disabling disease (EDSS > 3.5) (3.52 T 1.06 pg/ml) compared with those with mild disability (EDSS < 3.5) (3.3 T 0.8 pg/ ml, p = ns). CSF IL-15/Serum IL-15 ratio (C/S IL-15 ratio) was found lower in MS patients (0.79 T 0.25) compared with IND (1.06 T 0.35, p = 0.01) and not significantly different compared with NIND patients (0.90 T 0.28, p = 0.14) (Fig. 4).
5. Discussion In the present study serum IL-15 levels were elevated in the MS group compared with IND or NIND control patients (Fig. 1). The increase of IL-15 concentrations was not restricted to RRMS patients but was expanded to all the clinical subtypes of the disease. In two recent studies the elevation of IL-15-positive peripheral blood mononuclear cells (PBMNC) was restricted or was most pronounced to patients with chronic progressive MS [19,20]. In the subgroup of our patients with the PP form of the
10 Mean
+SD
1,5
9
Mean
1,4 8
1,3 1,2
c/s IL-15
s IL-15
7 6 5
1,1 1,0
4
0,9
3
0,8 0,7
2 1
+SD
0,6 RR
IDS
SP
PP
Fig. 2. Distribution of IL-15 levels in serum samples of patients with RRMS, SPMS, PPMS, and IDS. RRMS = relapsing – remitting multiple sclerosis, SPMS = secondary progressive multiple sclerosis, PPMS = primary progressive multiple sclerosis, IDS = isolated demyelinating syndrome.
0,5
MS
IND
NIND
Fig. 4. Distribution of c/s IL-15 ratio in MS, IND and NIND patients. c/s IL-15 = CSF/Serum IL-15, MS = multiple sclerosis, IND = inflammatory neurological diseases, NIND = non-inflammatory neurological diseases.
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M. Rentzos et al. / Journal of the Neurological Sciences 241 (2006) 25 – 29
disease serum IL-15 levels were found significantly different compared to all other types of the disease (Fig. 2). This might be due to the very elevated serum IL-15 value (12.9 pg/ml) of one patient with PPMS. However, it might be the result of the different immunogenetic profile of the PPMS regarding the other clinical forms of the disease. This is supported by the lower IgG index compared to RRMS, the lack of change on conventional imaging in the presence of clinical deterioration and the negative effect of immunomodulating or immunosuppressing treatment [24,25]. To determine whether IL-15 may or may not act more intensely in chronic progressive compared to RRMS more studies in a greater number of patients will be needed. In our study, serum IL-15 levels in the subgroup of patients with IDS were elevated compared to IND and NIND patients but did not show significant difference compared to clinical subtypes of definite MS (Fig. 2). Patients with IDS are at high risk for the development of definite MS in the future. Our findings lead to the view that IL-15 may be active from the beginning, years before the definite diagnosis of the disease. IL-15 serum levels were higher in patients with active gadolinium enhancing lesions compared to patients without enhancing MRI lesions [18]. Increased levels of IL-15 mRNA have also been detected in patients with a relapse compared with patients in a stable phase of the disease and healthy controls [10]. An increased expression of IL-15 mRNA has also been found in MS active plaques [1]. In the contrary comparison of serum IL-15 levels in our MS patients according to MRI activity showed no significant difference. No correlation with duration of the disease and EDSS has also been found. Our results are in agreement with the conclusions of other studies in which no correlation between clinical parameters and numbers of IL-15-positive PBMNC or IL-15 mRNA levels was observed [10,19]. CSF IL-15 levels in MS patients were higher compared to control patients with IND or NIND (Fig. 3). To our knowledge this is the first study in which there was a statistically significant difference of CSF IL-15 values among studied groups of patients. In another study most patients had non-detectable CSF IL-15 values [19]. This might be due to the lower limit of detection (16 pg/ml) of ELISA used. The rest of patients had no higher levels compared to other control groups. In this point a critical question is which is the origin of increased IL-15 levels in the CSF. Is high IL-15 a result of systemic production or is there an intrathecal production? To deal with this problem we introduced C/S IL-15 ratio as an index of possible intrathecal IL-15 production. This ratio was found lower in MS patients compared with the IND group and was not different compared with the NIND group (Fig. 4). IND group includes mostly patients with GBS in which a systemic activation of B cells and passive passage of antibodies through damaged blood – brain barrier into the
CNS compartment has been implicated [26]. Pashenkov et al. [19] have not detected IL-15 protein produced by CSF mononuclear cells immunocytochemically. Using ELISA to detect secreted IL-15, they found detectable levels in CSF samples from only 4 of 14 patients with RRMS, 2 of 8 patients with chronic progressive MS and 2 of 9 patients with other neurological diseases studied as control group. Their patients with other neurological diseases had lower CSF levels of IL-15 compared to serum, whereas patients with RR or chronic progressive MS did not. The authors’ conclusion is that according their results MS patients may have increased intrathecal production of IL-15. We believe that this is a very limited number of CSF samples where IL15 values have been detected to suggest that patients with MS may have increased intrathecal production of IL-15. Our results showed that systemic activation might be the source of high CSF IL-15 levels in MS patients. This does not exclude the possible IL-15 production by astrocytes, microglia or activated macrophages within CNS parenchyma. It is not clear how exactly IL-15 acts in the immune reactions of the disease. Involvement of IL-15 has been demonstrated in many autoimmune and infectious diseases such as rheumatoid arthritis, sarcoidosis and other diseases where cellular immunity and systemic activation are implicated [19,27,28]. Systemic origin of IL-15 might be the result of infections triggering relapses of the disease [29]. A general immune dysregulation associated with MS has also been reported recently [30,31]. Our findings suggest a possible role of IL-15 in the development of MS. There is little information about the involvement of this protein in the development of relapses, in blood – brain barrier permeability disturbances and in pathogenetic mechanisms involved in demyelination and axonal damage which are responsible for the increasing disability of MS. Further in vitro and in vivo studies are necessary to elucidate the contribution of IL-15 in inflammatory processes of the disease. Future treatment modalities focusing IL-15 as target might be added to other immunomodulatory drugs in order to ameliorate quality of life in MS patients. References [1] Baranzini S, Elfstrom C, Chang SY, et al. Transcriptional analysis of multiple sclerosis brain lesions reveals a complex pattern of cytokine expression. J Immunol 2000;165:6576 – 82. [2] Brosnan C, Raine C. Mechanisms of immune injury in multiple sclerosis. Brain Pathol 1996;6:243 – 57. [3] Xiao BG, Link H. Immune regulation within the central nervous system. J Neurol Sci 1998;157:1 – 12. [4] Beck J, Rondot P, Kirchner H, Wietzerbin J. Increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: do cytokines trigger off exacerbations? Acta Neurol Scand 1998;78:318 – 23. [5] Cannella B, Raine CS. The adhesion molecule and cytokine profile of multiple sclerosis lesions. Ann Neurol 1995;37:424 – 35. [6] Rieckmann P, Albrecht M, Kitze B, et al. Tumor necrosis factor-a messenger RNA expression in patients with relapsing – remitting
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multiple sclerosis is associated with disease activity. Ann Neurol 1995;37:82 – 8. Rieckmann P, Albrecht M, Kitze B, et al. Cytokine mRNA levels in mononuclear blood cells from patients with multiple sclerosis. Neurology 1994;44:1523 – 6. Link H. The cytokine storm in multiple sclerosis. Mult Scler 1998;4:12 – 5. Gayo A, Mozo L, Suarez A, Tunon A, Lahoz C, Gutierez C. Glucocorticoids increase IL-10 expression in multiple sclerosis patients with acute relapse. J Neuroimmunol 1998;85:122 – 30. Blanco – Jerez C, Plaza JF, Masjuan J, Orensanz LM, AlvarezCermeno JC. Increased levels of IL-15 mRNA in relapsing – remitting multiple sclerosis attacks. J Neuroimmunol 2002;128:90 – 4. Giri J, Kumaki S, Ahdie M, et al. Identification and cloning of a novel IL-15 binding protein that is structurally related to the a-chain of the IL-2 receptor. EMBO J 1995;14:3654 – 63. Grabstein KH, Eisenmann J, Shanebeck K, et al. Cloning of a T cell growth factor that interacts with the beta chain of the interleukin-2 receptor. Science 1994;264:965 – 8. Jonuleit H, Wiedemann K, Muller G. Induction of IL-15 messenger RNA and protein in human blood-derived dendritic cells. J Immunol 1997;160:2610 – 5. Lee YB, Satoh J, Walker DG, Kim SU. Interleukin-15 gene expression in human astrocytes and microglia in culture. Neuroreport 1996; 7:1062 – 6. Doherty TM, Seder RA, Sher A. Induction and regulation of IL-15 expression in murine macrophages. J Immunol 1996;156:735 – 41. Carson WE, Giri JG, Lindemann Mjet, et al. IL-15 is a novel cytokine that activates human NK cells via components of IL-2 receptor. J Exp Med 1994;180:1395 – 405. Carson WE, Ross ME, Baiocchi Ra, et al. Endogenous production of IL-15 by activated human mcrophages is critical for optimal production of IFN-g by NK cells in vitro. J Clin Invest 1995; 96:2578 – 89. Losy J, Niezgoda A, Zaremba J. IL-15 is elevated in sera of patients with relapsing – remitting multiple sclerosis. Folia Neuropathol 2002;40:151 – 3. Pashenkov M, Mustafa M, Kivisakk P, Link H. Levels of interleukin15 expressing blood mononuclear cells are elevated in multiple sclerosis. Scand J Immunol 1999;50:302 – 8.
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[20] Kivisakk P, Matusevicius HE, Soderstrom M, Fredrikson S, Link H. IL-15 mRNA is up-regulated in blood and cerebrospinal fluid mononuclear cells in multiple sclerosis. Clin Exp Immunol 1998; 111:193 – 8. [21] McDonald WI, Compston A, Edan G, et al. Recommended diagnostic criteria for multiple sclerosis: guidelines from the international panel on the diagnosis of multiple sclerosis. Ann Neurol 2001;50:121 – 7. [22] Jacobs LD, Beck RW, Simon JH, et al. Intramuscular interferon beta1a therapy initiated during a first demyelinating event in multiple sclerosis. N Engl J Med 2000;357:898 – 904. [23] Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an Expanded Disability Status Scale (EDSS). Neurology 1983;33: 1444 – 52. [24] Thompson AJ, Polman CH, Miller DH, et al. Primary progressive multiple sclerosis. Brain 1997;120:1085 – 96. [25] Michalopoulou M, Nikolaou C, Tavernarakis A, et al. Soluble interleukin-6 receptor (sIL-6R) in cerebrospinal fluid of patients with inflammatory and non inflammatory neurological diseases. Immunol Lett 2004;94:183 – 9. [26] Andersson M, Alvarez-Germeno J, Bernardi G, et al. Cerebrospinal fluid in the diagnosis of multiple sclerosis: a consensus report. J Neurol Neurosurg Psychiatry 1994;57:897 – 902. [27] McInnes IB, al-Mughales J, Field M, et al. The role of interleukin-15 in T-cell migration and activation in rheumatoid arthritis. Nat Med 1996;2:175 – 82. [28] Agostini C, Trentin L, Facco M, et al. Role of IL-15, IL-2 and their receptors in the development of T cell alveolitis in pulmonary sarcoidosis. J Immunol 1997;157:910 – 8. [29] Zeman A, McLean B, Keir G, Luxton R, Sharief M, Thompson E. The significance of serum oligoclonal bands in neurological diseases. J Neurol Neurosurg Psychiatry 1993;56:32 – 5. [30] Rentzos M, Michalopoulou M, Gotosidis K, et al. Unusual association of multiple sclerosis and monoclonal gammopathy of undetermined significance: two case reports. Funct Neurol 2004;4(19):254 – 6. [31] Trefouret S, Azulay JP, Pouget J, Boucraut J, Serratrice G, et al. Late – onset multiple sclerosis and monoclonal gammopathy: a fortuitous association? Rev Neurol 1996;152:554 – 6.
IL-15 is elevated in serum and cerebrospinal fluid of patients with multiple sclerosis M. Rentzos a,*, C. Cambouri b, A. Rombos a, C. Nikolaou b, M. Anagnostouli a, A. Tsoutsou b, A. Dimitrakopoulos a, N. Triantafyllou a, D. Vassilopoulos a a
Department of Neurology, Aeginition Hospital-Athens Medical School, 72-74 Vas.So phias Av, Greece b Department of Biopathology-Immunology, Aeginition Hospital-Athens Medical School, Greece Received 11 May 2005; received in revised form 26 August 2005; accepted 5 October 2005 Available online 28 November 2005
Abstract Interleukin-15 (IL-15) is a novel proinflammatory cytokine having similar biological activities to IL-2 which is implicated in the pathogenesis of multiple sclerosis. It is produced by activated blood monocytes, macrophages and glial cells. There is little information about the involvement of IL-15 in the development of multiple sclerosis (MS). The objective of our study was to measure IL-15 serum and cerebrospinal fluid (CSF) levels in MS patients and to correlate serum and CSF IL-15 concentrations with clinical parameters of the disease. CSF IL-15/Serum IL-15 ratio (c/s IL-15 ratio) was introduced to assess the origin of elevated IL-15 levels. Materials and methods: We measured serum and CSF IL-15 levels in 52 patients with MS and 36 age and gender matched patients with inflammatory (IND) and non-inflammatory neurological diseases (NIND) studied as control groups. IL-15 levels were correlated with clinical parameters as duration, disability, MRI activity and clinical subtypes of the disease. Results: MS patients were found to have significantly higher serum IL-15 levels compared with IND ( p = 0.00016) and NIND patients ( p = 0.00045). Elevated levels of IL-15 were also found in CSF samples from MS patients compared with patients with IND ( p = 0.00034) and NIND ( p = 0.0003). Among MS subgroups there were no statistically different IL-15 serum and CSF concentrations. No significant correlation of serum and CSF IL-15 concentrations with MRI activity, disability assessed by EDSS score and duration of the disease were also found. C/S IL-15 ratio was found lower in MS patients compared with IND ( p = 0.01) and not significantly different compared with NIND patients ( p = 0.14) suggesting that systemic activation might be the source of high CSF IL-15 levels in MS patients. Conclusions: Our findings suggest a possible role of IL-15 in the immunopathogenetic mechanisms of MS. D 2005 Elsevier B.V. All rights reserved. Keywords: IL-15; Multiple sclerosis; Clinical parameters; Immunopathogenetic mechanisms
1. Introduction Multiple sclerosis (MS) is a demyelinating disease of the central nervous system (CNS). Its cause remains unknown, although there is evidence of a complex and multifactorial aetiology with an underlying genetic susceptibility associated with unknown environmental factors [1]. Lymphocyte activation, extravasation and infiltration into the CNS together with activation of potentially immunocompetent
* Corresponding author. Tel.: +30 210 7289200; fax: +30 210 7219860. E-mail address: [email protected] (M. Rentzos). 0022-510X/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2005.10.003
glial cells are considered to lead to myelin destruction [2,3]. Proinflammatory T-Helper (TH)-1 cytokines such as Tumor Necrosis Factor, Interferon-g and Interleukin-2 seem to be related to inflammation and tissue damage in MS [4 – 7]. In the contrary, TH2/TH3 cytokines may contribute to suppress disease activity and progression [8 –10]. IL-15 is a proinflammatory cytokine. Its three-dimensional structure is similar to that of IL-2, although they do not share a significant homology. Their receptors share h and g chains, but IL-15 receptor a-chain is unique [11]. IL15 is produced by activated blood monocytes, macrophages, dendritic cells and activated glial cells [12 – 16]. It promotes T cell proliferation, induction of cytolytic effector cells
26
M. Rentzos et al. / Journal of the Neurological Sciences 241 (2006) 25 – 29
including natural killer (NK) and cytotoxic cells and stimulates B-cell to proliferate and secrete immunoglobulins [6,17]. Little information is available on the exact role of IL-15 in the development of MS lesions [10,18 –20]. Our study is focused on IL-15 production in MS patients, as well as on possible correlations between serum and cerebrospinal fluid (CSF) IL-15 levels and clinical subtype of the disease, presence of active lesions in MRI, duration of the disease and disability status.
2. Materials and methods 2.1. Patients Serum samples were studied in 80 individuals comprising the following groups: (1) 52 patients with MS (25 men and 27 women, aged 18 to 68 years, mean 35); (2) 20 agematched patients with other non-inflammatory neurological diseases (NIND); 5 out of these had amyotrophic lateral sclerosis (ALS), 3 had dementia, 3 had Parkinson’s disease and 10 had epilepsy; (3) 16 age-matched patients with other inflammatory neurological diseases (IND); 12 out of these had Guillain –Barre` Syndrome (GBS) and 4 had Chronic Inflammatory Demyelinating Polyneuropathy (CIDP). The second and third groups were studied as a control group. All of MS patients had definite MS according to the criteria of McDonald et al. [21] except for 6 patients that had isolated demyelinating syndrome (IDS) and were at high risk for the development of multiple sclerosis. In IDS group, patients with optic neuritis, diplopia, hemiparesis and cerebellar ataxia were included. These patients presented the first demyelinating event and had two or more clinically silent brain lesions on MRI [22]. MS patients were scored by the Expanded Disability Status Scale (EDSS) [23]. Mean EDSS score was 3.0 T 1.4, range 1.5 – 7.5. They were divided into two groups: 32 patients had EDSS < 3.5 and 14 had EDSS > 3.5. The mean duration of the disease was 7 T 7.5, range 0.08– 43 years. 30 patients had duration of the disease <10 years and 16 > 10 years. Of the 46 patients with MS 29 had the relapsing –remitting (RR) type of the disease, 13 had the secondary progressive (SP), and 4 the primary progressive (PP) type of the disease. MS patients were also divided into two other groups: 9 with brain or spinal cord magnetic resonance imaging (MRI) activity and 37 without. MS patients were considered to have MRI activity when they had one or more enhancing lesions in T1-weighted spin-echo images after gadopentate dimeglumine (Gd-DTPA) injection. GdDTPA was given intravenously at a dose of 0.1 mmol/kg and about 15 min after contrast injection a T1-weighted sequence was repeated. Ethical consent has been obtained by all patients and controls for studying IL-15 serum and CSF levels. Serum samples were stored at 70 -C until assayed. Cerebrospinal fluid (CSF) samples of patients with
MS, NIND and IND were obtained by lumbar puncture. CSF was centrifuged to separate the cells and then frozen within 2 h in multiple aliquots at 70 -C. No patient with MS had received any immunomodulatory treatment for at least 3 months prior to study. 2.2. Serum and CSF IL-15 assays Serum and CSF concentrations of circulating IL-15 were measured by immunoassay using commercially available enzyme-like immunosorbent assay (ELISA) kits (R&D systems). Undiluted serum and CSF samples were added in duplicate to microtiter wells and assayed according to routine procedures. Optical densities were determined by means of a micro-ELISA reader (L.P 400, Pasteur diagnostics). Values were calculated by comparison with a standard curve that was generated with IL-15 standards. The limit of detection was 2 pg/ml.
3. Statistical analysis Results were expressed as mean T standard deviation. The Student’s t-test and the Mann – Whitney U-test were used, as appropriate, to compare titres of IL-15 between the examined study groups. Data are expressed as the mean T S.D. All comparisons were two-sided, with a pvalue of less than 0.05 used to indicate statistical significance. The statistical software used for this analysis was Statistica 6.0.
4. Results 4.1. Serum IL-15 levels Serum IL-15 levels were significantly elevated in the MS group (mean T standard deviation [S.D.], 4.2 T 1.36 pg/ml) compared with patients with IND (1.95 T 0.41 pg/ml; p = 0.00016) and NIND (2.96 T 1.13 pg/ml; p = 0.00045 (Fig. 1). Serum IL-15 levels were not significantly different in RRMS patients (4.13 T 0.61 pg/ml) compared with SPSM patients (4.26 T 0.73 pg/ml; p = 0.64) and IDS (4.13 T 0.45 pg/ml; p = 0.99) (Fig. 2). PPMS patients (5.56 T 3.6 pg/ml) had higher IL-15 values compared to patients with all other types of the disease (4.15 T 0.6 pg/ml; p = 0.018) (Fig. 2). Among MS patients, patients with MRI activity (mean T S.D., 3.97 T 0.57 pg/ml) had no significantly different serum IL-15 levels when compared with patients with no MRI activity (4.38 T 1.54 pg/ml, p = ns). IL-15 levels were not also different in patients with over 10 years duration of the disease (4.92 T 0.56 pg/ml) compared with those under 10 years (4.1 T 0.62 pg/ml, p = ns) or patients with more disabling disease (EDSS > 3.5) (5.00 T 2.64 pg/ml) compared with those with mild disability (EDSS < 3.5) (4.1 T 0.81 pg/ ml, p = ns).
M. Rentzos et al. / Journal of the Neurological Sciences 241 (2006) 25 – 29 6,0 5,5
Mean
+SD
5,0
c IL-15
s IL-15
4,5 4,0 3,5 3,0 2,5 2,0 1,5 1,0
MS
IND
NIND
Fig. 1. Distribution of IL-15 levels in serum samples of patients with MS, IND and NIND. MS = multiple sclerosis, IND = inflammatory neurological diseases, NIND = non-inflammatory neurological diseases.
4.2. CSF IL-15 levels Elevated levels of IL-15 were found in CSF samples from patients with MS (mean T S.D., 3.27 T 0.91 pg/ml) compared with IND (2 T 0.47 pg/ml; p = 0.00034) and NIND patients (2.43 T 0.49 pg/ml; p = 0.00029) (Fig. 3). CSF IL-15 levels were not significantly different in RRMS patients (3.34 T 0.9 pg/ml) compared with SPMS (3.31 T1.03 pg/ml; p = 0.93), IDS (3.5 T 0.84 pg/ml; p = 0.7) and PPMS patients (3.43 T 0.8 pg/ml; p = 0.83). Among MS patients, patients with MRI activity (mean T S.D., 3.12 T 1.22 pg/ml) had no significantly different CSF IL-15 levels when compared with patients with no MRI activity (3.38 T 0.88 pg/ml, p = ns). IL-15 levels were not also different in patients with over 10 years duration of the disease (3.6 T 0.71 pg/ml) compared with those under 10 years (3.21 T 0.48 pg/ml, p = ns) or patients with more
4,4 4,2 4,0 3,8 3,6 3,4 3,2 3,0 2,8 2,6 2,4 2,2 2,0 1,8 1,6 1,4
27
Mean
MS
IND
+SD
NIND
Fig. 3. Distribution of IL-15 levels in CSF samples of patients with MS, IND and NIND. MS = multiple sclerosis, IND = inflammatory neurological diseases, NIND = non-inflammatory neurological diseases.
disabling disease (EDSS > 3.5) (3.52 T 1.06 pg/ml) compared with those with mild disability (EDSS < 3.5) (3.3 T 0.8 pg/ ml, p = ns). CSF IL-15/Serum IL-15 ratio (C/S IL-15 ratio) was found lower in MS patients (0.79 T 0.25) compared with IND (1.06 T 0.35, p = 0.01) and not significantly different compared with NIND patients (0.90 T 0.28, p = 0.14) (Fig. 4).
5. Discussion In the present study serum IL-15 levels were elevated in the MS group compared with IND or NIND control patients (Fig. 1). The increase of IL-15 concentrations was not restricted to RRMS patients but was expanded to all the clinical subtypes of the disease. In two recent studies the elevation of IL-15-positive peripheral blood mononuclear cells (PBMNC) was restricted or was most pronounced to patients with chronic progressive MS [19,20]. In the subgroup of our patients with the PP form of the
10 Mean
+SD
1,5
9
Mean
1,4 8
1,3 1,2
c/s IL-15
s IL-15
7 6 5
1,1 1,0
4
0,9
3
0,8 0,7
2 1
+SD
0,6 RR
IDS
SP
PP
Fig. 2. Distribution of IL-15 levels in serum samples of patients with RRMS, SPMS, PPMS, and IDS. RRMS = relapsing – remitting multiple sclerosis, SPMS = secondary progressive multiple sclerosis, PPMS = primary progressive multiple sclerosis, IDS = isolated demyelinating syndrome.
0,5
MS
IND
NIND
Fig. 4. Distribution of c/s IL-15 ratio in MS, IND and NIND patients. c/s IL-15 = CSF/Serum IL-15, MS = multiple sclerosis, IND = inflammatory neurological diseases, NIND = non-inflammatory neurological diseases.
28
M. Rentzos et al. / Journal of the Neurological Sciences 241 (2006) 25 – 29
disease serum IL-15 levels were found significantly different compared to all other types of the disease (Fig. 2). This might be due to the very elevated serum IL-15 value (12.9 pg/ml) of one patient with PPMS. However, it might be the result of the different immunogenetic profile of the PPMS regarding the other clinical forms of the disease. This is supported by the lower IgG index compared to RRMS, the lack of change on conventional imaging in the presence of clinical deterioration and the negative effect of immunomodulating or immunosuppressing treatment [24,25]. To determine whether IL-15 may or may not act more intensely in chronic progressive compared to RRMS more studies in a greater number of patients will be needed. In our study, serum IL-15 levels in the subgroup of patients with IDS were elevated compared to IND and NIND patients but did not show significant difference compared to clinical subtypes of definite MS (Fig. 2). Patients with IDS are at high risk for the development of definite MS in the future. Our findings lead to the view that IL-15 may be active from the beginning, years before the definite diagnosis of the disease. IL-15 serum levels were higher in patients with active gadolinium enhancing lesions compared to patients without enhancing MRI lesions [18]. Increased levels of IL-15 mRNA have also been detected in patients with a relapse compared with patients in a stable phase of the disease and healthy controls [10]. An increased expression of IL-15 mRNA has also been found in MS active plaques [1]. In the contrary comparison of serum IL-15 levels in our MS patients according to MRI activity showed no significant difference. No correlation with duration of the disease and EDSS has also been found. Our results are in agreement with the conclusions of other studies in which no correlation between clinical parameters and numbers of IL-15-positive PBMNC or IL-15 mRNA levels was observed [10,19]. CSF IL-15 levels in MS patients were higher compared to control patients with IND or NIND (Fig. 3). To our knowledge this is the first study in which there was a statistically significant difference of CSF IL-15 values among studied groups of patients. In another study most patients had non-detectable CSF IL-15 values [19]. This might be due to the lower limit of detection (16 pg/ml) of ELISA used. The rest of patients had no higher levels compared to other control groups. In this point a critical question is which is the origin of increased IL-15 levels in the CSF. Is high IL-15 a result of systemic production or is there an intrathecal production? To deal with this problem we introduced C/S IL-15 ratio as an index of possible intrathecal IL-15 production. This ratio was found lower in MS patients compared with the IND group and was not different compared with the NIND group (Fig. 4). IND group includes mostly patients with GBS in which a systemic activation of B cells and passive passage of antibodies through damaged blood – brain barrier into the
CNS compartment has been implicated [26]. Pashenkov et al. [19] have not detected IL-15 protein produced by CSF mononuclear cells immunocytochemically. Using ELISA to detect secreted IL-15, they found detectable levels in CSF samples from only 4 of 14 patients with RRMS, 2 of 8 patients with chronic progressive MS and 2 of 9 patients with other neurological diseases studied as control group. Their patients with other neurological diseases had lower CSF levels of IL-15 compared to serum, whereas patients with RR or chronic progressive MS did not. The authors’ conclusion is that according their results MS patients may have increased intrathecal production of IL-15. We believe that this is a very limited number of CSF samples where IL15 values have been detected to suggest that patients with MS may have increased intrathecal production of IL-15. Our results showed that systemic activation might be the source of high CSF IL-15 levels in MS patients. This does not exclude the possible IL-15 production by astrocytes, microglia or activated macrophages within CNS parenchyma. It is not clear how exactly IL-15 acts in the immune reactions of the disease. Involvement of IL-15 has been demonstrated in many autoimmune and infectious diseases such as rheumatoid arthritis, sarcoidosis and other diseases where cellular immunity and systemic activation are implicated [19,27,28]. Systemic origin of IL-15 might be the result of infections triggering relapses of the disease [29]. A general immune dysregulation associated with MS has also been reported recently [30,31]. Our findings suggest a possible role of IL-15 in the development of MS. There is little information about the involvement of this protein in the development of relapses, in blood – brain barrier permeability disturbances and in pathogenetic mechanisms involved in demyelination and axonal damage which are responsible for the increasing disability of MS. Further in vitro and in vivo studies are necessary to elucidate the contribution of IL-15 in inflammatory processes of the disease. Future treatment modalities focusing IL-15 as target might be added to other immunomodulatory drugs in order to ameliorate quality of life in MS patients. References [1] Baranzini S, Elfstrom C, Chang SY, et al. Transcriptional analysis of multiple sclerosis brain lesions reveals a complex pattern of cytokine expression. J Immunol 2000;165:6576 – 82. [2] Brosnan C, Raine C. Mechanisms of immune injury in multiple sclerosis. Brain Pathol 1996;6:243 – 57. [3] Xiao BG, Link H. Immune regulation within the central nervous system. J Neurol Sci 1998;157:1 – 12. [4] Beck J, Rondot P, Kirchner H, Wietzerbin J. Increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: do cytokines trigger off exacerbations? Acta Neurol Scand 1998;78:318 – 23. [5] Cannella B, Raine CS. The adhesion molecule and cytokine profile of multiple sclerosis lesions. Ann Neurol 1995;37:424 – 35. [6] Rieckmann P, Albrecht M, Kitze B, et al. Tumor necrosis factor-a messenger RNA expression in patients with relapsing – remitting
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