Journal of Neuroimmunology 271 (2014) 56–59
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Levels of soluble TNF-RII are increased in serum of patients with primary progressive multiple sclerosis Nicolás Fissolo ⁎, Ester Cantó, Angela Vidal-Jordana, Joaquín Castilló, Xavier Montalban, Manuel Comabella Department of Neurology–Neuroimmunology, Centre d'Esclerosi Múltiple de Catalunya, Cemcat, Institut de Recerca Vall d'Hebron, Hospital Universitari Vall d'Hebron (HUVH), Barcelona, Spain
a r t i c l e
i n f o
Article history: Received 26 February 2014 Received in revised form 3 April 2014 Accepted 8 April 2014 Keywords: Primary progressive multiple sclerosis Tumor necrosis factor receptor II Disability progression
a b s t r a c t The levels of soluble tumor necrosis factor receptor II (sTNF-RII) were determined in serum of 161 untreated multiple sclerosis (MS) patients with different clinical forms and 46 healthy controls (HC) by ELISA. Our results show that serum sTNF-RII levels were significantly increased in patients with primary progressive MS (PPMS) compared with other MS forms and HC. Although sTNF-RII levels significantly increased over a 2-year follow-up period in a subgroup of PPMS patients, they could not discriminate between patients with and without disability progression. Additional studies are needed to further implicate sTNF-RII in patients with PPMS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Tumor necrosis factor (TNF) is a pleiotropic and highly regulated proinflammatory cytokine with roles in important processes such as inflammation, cell proliferation, and apoptosis (Wallach et al., 1999). Its aberrant production has been reported to promote a wide range of chronic inflammatory pathologies, including neurological disorders (Baker et al., 1994; Korner et al., 1997; Akassoglou et al., 1998). TNF activity is mediated by two ubiquitously expressed transmembrane receptors, TNF-receptor type I (TNF-RI) and type II (TNF-RII) (Beutler and Van Huffel, 1994; Tracey and Cerami, 1994). Soluble TNF-RII (sTNF-RII) is one of the two soluble TNF-α receptors found in human biological fluids, which are released from the cell surface by proteolytic cleavage (shedding) of the transmembrane TNF-RII (Porteu and Nathan, 1990). sTNF-RII has been suggested to bind and neutralize TNF-α and TNF-β, thus potentially inhibiting their proinflammatory effects (Seckinger et al., 1989; Mohler et al., 1993). TNF-α has long been involved in the pathogenesis of multiple sclerosis (MS) (Selmaj and Raine, 1988; Selmaj et al., 1991; Caminero et al., 2011), and cerebrospinal fluid (CSF) and serum levels of this cytokine were found to correlate with MS disease progression (Sharief and Hentges, 1991; Rieckmann et al., 1994). In this line, a recently published paper by Hagman et al. (2011) identified TNF-α, among other cytokines, as a candidate biomarker of disease activity and progression in patients
with primary progressive MS (PPMS). Whereas most studies have determined the levels of TNF-α in serum of MS patients, the concentrations of their soluble receptors have received little attention. A recent publication showed the presence of a soluble isoform of the TNF-RI associated with a risk allele for MS located in the TNFRSF1A gene (Gregory et al., 2012). Based on these findings, the purpose of the present study was to investigate the role of the other TNF receptor, TNF-RII, in MS by measuring the serum levels of this receptor in patients with relapsing and progressive clinical forms of MS, and to correlate sTNF-RII levels with clinical and radiological variables.
2. Materials and methods 2.1. Patients Forty six healthy controls and 161 patients with clinically definite MS who had not received treatment with immunomodulatory therapy were included in the study. The study was approved by the Ethics Committee of Vall d'Hebron University Hospital and all the subjects involved in the study gave written informed consent. Demographic and baseline clinical characteristics of MS patients and healthy controls are summarized in Table 1.
2.2. Serum sTNF-RII levels and MS clinical course ⁎ Corresponding author at: Unitat de Neuroimmunologia Clínica, Cemcat, Hospital Universitari Vall d'Hebron. Pg. Vall d'Hebron 119-129 08035 Barcelona, Spain. Tel.: +34 932746843; fax: +34 932746084. E-mail address: nicolas.fi[email protected] (N. Fissolo).
http://dx.doi.org/10.1016/j.jneuroim.2014.04.001 0165-5728/© 2014 Elsevier B.V. All rights reserved.
To investigate the serum levels of sTNF-RII in patients with different clinical forms of MS, the disease course of individual patients was classified as relapsing–remitting (RRMS, n = 49), secondary progressive
N. Fissolo et al. / Journal of Neuroimmunology 271 (2014) 56–59
57
Table 1 Demographic and baseline clinical characteristics of MS patients and HC. Characteristics
HC
RRMS
SPMS
PPMS
n Female/male (% women) Age (years)a Duration of disease (years)a EDSSb Number of relapses in the 2 previous yearsa
46 31/15 (67.4) 37.4 (10.6) – – –
49 35/14 (71.4) 37.5 (8.6) 7.8 (6.6) 2.2 (1.5–2.5) 2.0 (1.5)
39 28/11 (71.8) 47.7 (8.8) 14.7 (9.2) 4.8 (4.0–6.0) 0.8 (0.8)
73 35/38 (47.9) 50.6 (8.3) 11.4 (7.2) 5.3 (4.0–6.5) –
EDSS: expanded disability status scale; RRMS: relapsing–remitting multiple sclerosis; SPMS: secondary progressive multiple sclerosis; PPMS: primary progressive multiple sclerosis; HC: healthy controls. a Data are expressed as mean (SD). b Data are expressed as median (interquartile range).
(SPMS, n = 39), or primary progressive (PPMS, n = 73) according to the Lublin and Reingold classification (Lublin and Reingold, 1996).
3. Results 3.1. Serum sTNF-RII levels are elevated in patients with PPMS
To investigate a potential relationship between serum sTNF-RII levels and disability progression, a subgroup of PPMS patients (n = 24) was classified according to worsening of disability over a two-year period. Disability progression was defined as a confirmed increase in the EDSS score of at least 1 point if initial EDSS score was lower than 5.5, and of at least 0.5 points if initial EDSS score was equal or higher than 5.5 during the follow-up period. Serum levels of sTNF-RII were measured at baseline and after two years of follow-up in PPMS patients with disability progression (n = 11) and without disability progression (n = 13). 2.4. Determination of serum levels of sTNF-RII by enzyme-linked immunosorbent assay Peripheral blood was collected by standard venipuncture and serum was prepared after centrifugation of the clotted blood and stored frozen at − 80 °C until used. Serum levels of sTNF-RII were determined by a sandwich enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Human sTNF RII/TNFRSF1B Immunoassay, R&D), according the manufacturer's protocol. Samples were diluted at a 1:50 ratio by dilution buffer and added to ELISA plates to measure the sTNF-RII content in the serum. Plates were analyzed at OD 450 nm using an ELx800 Absorbance Microplate Reader (BioTek, VT, USA) and calculated using Gen5 Data Analysis Software (BioTek, VT, USA). All assays were performed in duplicate and samples with variation coefficients higher than 10% were repeated. The intra-assay and inter-assay coefficients of variation were 2.5% and 10% respectively. 2.5. Statistical analysis Statistical analysis was performed by using the SPSS 17.0 package (SPSS Inc, Chicago, IL) for MS-Windows. The ANOVA test was first used to analyze differences in serum sTNF-RII levels between MS patients with different clinical forms and healthy controls. An unpaired Student's t test was then used to test for significant differences in serum sTNF-RII levels between PPMS patients, MS patients with other clinical forms of the disease (RRMS and SPMS), and healthy controls. A paired Student's t-test was used to test for significant differences in serum sTNF-RII levels between PPMS patients at baseline and after two years of follow-up. Linear associations between serum sTNF-RII levels and clinical and radiological variables were assessed by the Pearson or Spearman rank correlation coefficients depending on the applicability conditions. Descriptive data are presented as mean values [standard deviation (SD)] unless otherwise stated.
Serum sTNF-RII levels were analyzed in serum samples from healthy controls and MS patients with different clinical forms of MS. As shown in Fig. 1, serum sTNF-RII levels were significantly increased in PPMS patients compared with RRMS patients (p = 0.005), SPMS patients (p = 0.047) and healthy controls (p = 0.018). 3.2. Levels of serum sTNF-RII are not associated with disability progression in PPMS patients We next investigated whether serum sTNF-RII levels changed over time in a subgroup of 24 PPMS patients. As shown in Fig. 2A, comparisons of sTNF-RII levels over the 24-month follow-up period revealed a statistically significant increase in protein levels at 24 months versus the baseline (p = 3.8 × 10−7). We further analyzed whether changes in serum sTNF-RII during the follow-up period could discriminate between patients with and without disability progression. Demographic
p=0.018 p=0.005 5000
p=0.047
4000
sTNF-RII (pg/ml)
2.3. Serum sTNF-RII levels and disability progression
3000
2000
1000
0 HC
RR
(N=46)
(N=49)
SP
(N=39)
PP
(N=73)
Fig. 1. Comparison of serum sTNF-RII levels between MS patients with different clinical forms of the disease and healthy controls. Box plots showing serum sTNF-RII levels in MS and healthy controls. sTNF-RII levels were determined by ELISA as described in Materials and methods. The levels of the soluble receptor are increased in PPMS patients compared with RRMS patients (median values: 2928 pg/ml in PPMS vs. 2594 pg/ml in RRMS; p = 0.005), SPMS patients (median: 2731 pg/ml; p = 0.047) and healthy controls (median: 2661 pg/ml; p = 0.018). Boxes represent the 90th and 10th percentiles divided horizontally by the median. Whiskers are drawn to the nearest value not beyond a standard span from the 90th and 10th percentiles. RR: patients with relapsing–remitting MS. PP: patients with primary progressive MS. SP: patients with secondary progressive MS. HC: healthy controls.
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N. Fissolo et al. / Journal of Neuroimmunology 271 (2014) 56–59
A
4. Discussion
7000
p=3.8x10-7
sTNF-RII (pg/ml)
6000 5000 4000 3000 2000 1000 0
B
7000
0
24
(N=24)
(N=24)
p=4.2x10-4
p=4.9x10-4
sTNF-RII (pg/ml)
6000 5000 4000 3000 2000 1000 0 24
0
no progression (N=13)
0
24
progression (N=11)
Fig. 2. Changes in serum sTNF-RII levels over a 24-month follow-up in PPMS patients. Box plots showing the serum sTNF-RII levels in PPMS patients (n = 24) at baseline and after 24 months of follow-up. sTNF-RII levels were determined by ELISA. (A) Levels of sTNF-RII are increased over time in the whole group of PPMS patients (p = 3.8 × 10−7). (B) In PPMS patients classified by disability progression, sTNF-RII levels were significantly elevated in both groups of patients (p = 4.9 × 10−4 in patients with progression and p = 4.2 × 10−4 in patients without progression). Boxes represent the 90th and 10th percentiles divided horizontally by the median. Whiskers are drawn to the nearest value not beyond a standard span from the 90th and 10th percentiles.
The present study was designed to investigate the serum levels of one of the soluble receptors of TNF-α, sTNF-RII, in different clinical forms of MS. Our results show a selective increase of sTNF-RII levels in PPMS patients compared with patients with other clinical forms of the disease and healthy controls. In addition, we observed that in this group of patients sTNF-RII levels tended to increase over time, although this finding was irrespective of disability progression. Finally, in PPMS patients we observed a lack of association between radiological variables, measuring inflammation and atrophy, and serum levels of sTNF-RII probably indicating that the levels of this soluble receptor may not reflect the whole extent of CNS damage. Studies over the past years suggest that soluble TNF receptors may enhance or inhibit TNF activity depending on their concentration (Aderka et al., 1992; Mohler et al., 1993). The pro-inflammatory cytokine TNF-α has been associated with MS disease activity and implicated in axon degeneration (Rieckmann et al., 1995; Siebert and Bruck, 2003). On the other hand, TNF-α has also potent anti-inflammatory properties (Liu et al., 1998), and several studies using anti-TNF therapy in MS patients have shown to increase disease activity (The Lenercept Multiple Sclerosis Study Group, 1999). These pro-inflammatory and antiinflammatory properties of TNF-α make it difficult to hypothesize whether changes in serum levels of sTNF-RII will be beneficial or detrimental in MS. In our study, the selective increase in sTNF-RII serum levels in PPMS patients suggests a role of the receptor in this particular progressive form of MS, since sTNF-RII levels were not elevated in the more inflammatory but also progressive form of SPMS. However, as outlined above, sTNF-RII did not correlate with disability progression or radiological measures of atrophy. Based on these findings, further studies will be required to elucidate the precise role of sTNF-RII in patients with PPMS. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jneuroim.2014.04.001. Acknowledgments The authors thank the “Red Española de Esclerosis Múltiple (REEM)” sponsored by the FEDER-FIS and the “Ajuts per donar Suport als Grups de Recerca de Catalunya”, sponsored by the “Agència de Gestió d'Ajuts Universitaris i de Recerca” (AGAUR), Generalitat de Catalunya, Spain. References
and baseline clinical characteristics were comparable between both groups of patients (Supplementary Table 1). As illustrated in Fig. 2B, serum sTNF-RII levels were similarly and significantly increased in PPMS patients with disability progression (p = 4.9 × 10− 4) and patients without disability progression (p = 4.2 × 10−4).
3.3. Correlations between serum sTNF-RII levels and clinical and radiological variables No significant correlations were found between serum TNF-RII levels in patients with different clinical forms of MS and clinical variables such as disease duration, number of relapses in the previous 2 years, and EDSS score at the time of blood drawing (correlation coefficient range: from −0.144 to 0.251). Similarly, no statistically significant correlations were observed between sTNF-RII levels in PPMS patients and baseline radiological variables such as T2-weighted lesion load, T1-weighted lesion load, brain parenchymal fraction, and spinal cord area (correlation coefficient range: 0.018–0.090).
Aderka, D., Engelmann, H., Maor, Y., Brakebusch, C., Wallach, D., 1992. Stabilization of the bioactivity of tumor necrosis factor by its soluble receptors. J. Exp. Med. 175, 323–329. Akassoglou, K., Bauer, J., Kassiotis, G., Pasparakis, M., Lassmann, H., Kollias, G., Probert, L., 1998. Oligodendrocyte apoptosis and primary demyelination induced by local TNF/ p55TNF receptor signaling in the central nervous system of transgenic mice: models for multiple sclerosis with primary oligodendrogliopathy. Am. J. Pathol. 153, 801–813. Baker, D., Butler, D., Scallon, B.J., O'Neill, J.K., Turk, J.L., Feldmann, M., 1994. Control of established experimental allergic encephalomyelitis by inhibition of tumor necrosis factor (TNF) activity within the central nervous system using monoclonal antibodies and TNF receptor–immunoglobulin fusion proteins. Eur. J. Immunol. 24, 2040–2048. Beutler, B., Van Huffel, C., 1994. An evolutionary and functional approach to the TNF receptor/ligand family. Ann. N. Y. Acad. Sci. 730, 118–133. Caminero, A., Comabella, M., Montalban, X., 2011. Tumor necrosis factor alpha (TNF-alpha), anti-TNF-alpha and demyelination revisited: an ongoing story. J. Neuroimmunol. 234, 1–6. Gregory, A., Dendrou, C., Attfield, K., Haghikia, A., Xifara, D., Butter, F., Poschmann, G., Kaur, G., Lambert, L., Leach, O., Prömel, S., Punwani, D., Felce, J., Davis, S., Gold, R., Nielsen, F., Siegel, R., Mann, M., Bell, J., McVean, G., Fugger, L., 2012. TNF receptor 1 genetic risk mirrors outcome of anti-TNF therapy in multiple sclerosis. Nature 488 (7412), 508–511. Hagman, S., Raunio, M., Rossi, M., Dastidar, P., Elovaara, I., 2011. Disease-associated inflammatory biomarker profiles in blood in different subtypes of multiple sclerosis: prospective clinical and MRI follow-up study. J. Neuroimmunol. 234, 141–147. Korner, H., Riminton, D.S., Strickland, D.H., Lemckert, F.A., Pollard, J.D., Sedgwick, J.D., 1997. Critical points of tumor necrosis factor action in central nervous system autoimmune inflammation defined by gene targeting. J. Exp. Med. 186, 1585–1590.
N. Fissolo et al. / Journal of Neuroimmunology 271 (2014) 56–59 Liu, J., Marino, M.W., Wong, G., Grail, D., Dunn, A., Bettadapura, J., Slavin, A.J., Old, L., Bernard, C.C., 1998. TNF is a potent anti-inflammatory cytokine in autoimmunemediated demyelination. Nat. Med. 4, 78–83. Lublin, F.D., Reingold, S.C., 1996. Defining the clinical course of multiple sclerosis: results of an international survey. National Multiple Sclerosis Society (USA) Advisory Committee on Clinical Trials of New Agents in Multiple Sclerosis. Neurology 46, 907–911. Mohler, K.M., Torrance, D.S., Smith, C.A., Goodwin, R.G., Stremler, K.E., Fung, V.P., Madani, H., Widmer, M.B., 1993. Soluble tumor necrosis factor (TNF) receptors are effective therapeutic agents in lethal endotoxemia and function simultaneously as both TNF carriers and TNF antagonists. J. Immunol. 151, 1548–1561. Porteu, F., Nathan, C., 1990. Shedding of tumor necrosis factor receptors by activated human neutrophils. J. Exp. Med. 172, 599–607. Rieckmann, P., Albrecht, M., Kitze, B., Weber, T., Tumani, H., Broocks, A., Luer, W., Poser, S., 1994. Cytokine mRNA levels in mononuclear blood cells from patients with multiple sclerosis. Neurology 44, 1523–1526. Rieckmann, P., Albrecht, M., Kitze, B., Weber, T., Tumani, H., Broocks, A., Luer, W., Helwig, A., Poser, S., 1995. Tumor necrosis factor-alpha messenger RNA expression in patients with relapsing–remitting multiple sclerosis is associated with disease activity. Ann. Neurol. 37, 82–88.
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Seckinger, P., Isaaz, S., Dayer, J.M., 1989. Purification and biologic characterization of a specific tumor necrosis factor alpha inhibitor. J. Biol. Chem. 264, 11966–11973. Selmaj, K.W., Raine, C.S., 1988. Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro. Ann. Neurol. 23, 339–346. Selmaj, K., Raine, C.S., Cannella, B., Brosnan, C.F., 1991. Identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions. J. Clin. Invest. 87, 949–954. Sharief, M.K., Hentges, R., 1991. Association between tumor necrosis factor-alpha and disease progression in patients with multiple sclerosis. N. Engl. J. Med. 325, 467–472. Siebert, H., Bruck, W., 2003. The role of cytokines and adhesion molecules in axon degeneration after peripheral nerve axotomy: a study in different knockout mice. Brain Res. 960, 152–156. The Lenercept Multiple Sclerosis Study Group, 1999. TNF neutralization in MS: results of a randomized, placebo-controlled multicenter study. The Lenercept Multiple Sclerosis Study Group and the University of British Columbia MS/MRI Analysis Group. Neurology 53, 457–465. Tracey, K.J., Cerami, A., 1994. Tumor necrosis factor: a pleiotropic cytokine and therapeutic target. Annu. Rev. Med. 45, 491–503. Wallach, D., Varfolomeev, E.E., Malinin, N.L., Goltsev, Y.V., Kovalenko, A.V., Boldin, M.P., 1999. Tumor necrosis factor receptor and Fas signaling mechanisms. Annu. Rev. Immunol. 17, 331–367.
Contents lists available at ScienceDirect
Journal of Neuroimmunology journal homepage: www.elsevier.com/locate/jneuroim
Short communication
Levels of soluble TNF-RII are increased in serum of patients with primary progressive multiple sclerosis Nicolás Fissolo ⁎, Ester Cantó, Angela Vidal-Jordana, Joaquín Castilló, Xavier Montalban, Manuel Comabella Department of Neurology–Neuroimmunology, Centre d'Esclerosi Múltiple de Catalunya, Cemcat, Institut de Recerca Vall d'Hebron, Hospital Universitari Vall d'Hebron (HUVH), Barcelona, Spain
a r t i c l e
i n f o
Article history: Received 26 February 2014 Received in revised form 3 April 2014 Accepted 8 April 2014 Keywords: Primary progressive multiple sclerosis Tumor necrosis factor receptor II Disability progression
a b s t r a c t The levels of soluble tumor necrosis factor receptor II (sTNF-RII) were determined in serum of 161 untreated multiple sclerosis (MS) patients with different clinical forms and 46 healthy controls (HC) by ELISA. Our results show that serum sTNF-RII levels were significantly increased in patients with primary progressive MS (PPMS) compared with other MS forms and HC. Although sTNF-RII levels significantly increased over a 2-year follow-up period in a subgroup of PPMS patients, they could not discriminate between patients with and without disability progression. Additional studies are needed to further implicate sTNF-RII in patients with PPMS. © 2014 Elsevier B.V. All rights reserved.
1. Introduction Tumor necrosis factor (TNF) is a pleiotropic and highly regulated proinflammatory cytokine with roles in important processes such as inflammation, cell proliferation, and apoptosis (Wallach et al., 1999). Its aberrant production has been reported to promote a wide range of chronic inflammatory pathologies, including neurological disorders (Baker et al., 1994; Korner et al., 1997; Akassoglou et al., 1998). TNF activity is mediated by two ubiquitously expressed transmembrane receptors, TNF-receptor type I (TNF-RI) and type II (TNF-RII) (Beutler and Van Huffel, 1994; Tracey and Cerami, 1994). Soluble TNF-RII (sTNF-RII) is one of the two soluble TNF-α receptors found in human biological fluids, which are released from the cell surface by proteolytic cleavage (shedding) of the transmembrane TNF-RII (Porteu and Nathan, 1990). sTNF-RII has been suggested to bind and neutralize TNF-α and TNF-β, thus potentially inhibiting their proinflammatory effects (Seckinger et al., 1989; Mohler et al., 1993). TNF-α has long been involved in the pathogenesis of multiple sclerosis (MS) (Selmaj and Raine, 1988; Selmaj et al., 1991; Caminero et al., 2011), and cerebrospinal fluid (CSF) and serum levels of this cytokine were found to correlate with MS disease progression (Sharief and Hentges, 1991; Rieckmann et al., 1994). In this line, a recently published paper by Hagman et al. (2011) identified TNF-α, among other cytokines, as a candidate biomarker of disease activity and progression in patients
with primary progressive MS (PPMS). Whereas most studies have determined the levels of TNF-α in serum of MS patients, the concentrations of their soluble receptors have received little attention. A recent publication showed the presence of a soluble isoform of the TNF-RI associated with a risk allele for MS located in the TNFRSF1A gene (Gregory et al., 2012). Based on these findings, the purpose of the present study was to investigate the role of the other TNF receptor, TNF-RII, in MS by measuring the serum levels of this receptor in patients with relapsing and progressive clinical forms of MS, and to correlate sTNF-RII levels with clinical and radiological variables.
2. Materials and methods 2.1. Patients Forty six healthy controls and 161 patients with clinically definite MS who had not received treatment with immunomodulatory therapy were included in the study. The study was approved by the Ethics Committee of Vall d'Hebron University Hospital and all the subjects involved in the study gave written informed consent. Demographic and baseline clinical characteristics of MS patients and healthy controls are summarized in Table 1.
2.2. Serum sTNF-RII levels and MS clinical course ⁎ Corresponding author at: Unitat de Neuroimmunologia Clínica, Cemcat, Hospital Universitari Vall d'Hebron. Pg. Vall d'Hebron 119-129 08035 Barcelona, Spain. Tel.: +34 932746843; fax: +34 932746084. E-mail address: nicolas.fi[email protected] (N. Fissolo).
http://dx.doi.org/10.1016/j.jneuroim.2014.04.001 0165-5728/© 2014 Elsevier B.V. All rights reserved.
To investigate the serum levels of sTNF-RII in patients with different clinical forms of MS, the disease course of individual patients was classified as relapsing–remitting (RRMS, n = 49), secondary progressive
N. Fissolo et al. / Journal of Neuroimmunology 271 (2014) 56–59
57
Table 1 Demographic and baseline clinical characteristics of MS patients and HC. Characteristics
HC
RRMS
SPMS
PPMS
n Female/male (% women) Age (years)a Duration of disease (years)a EDSSb Number of relapses in the 2 previous yearsa
46 31/15 (67.4) 37.4 (10.6) – – –
49 35/14 (71.4) 37.5 (8.6) 7.8 (6.6) 2.2 (1.5–2.5) 2.0 (1.5)
39 28/11 (71.8) 47.7 (8.8) 14.7 (9.2) 4.8 (4.0–6.0) 0.8 (0.8)
73 35/38 (47.9) 50.6 (8.3) 11.4 (7.2) 5.3 (4.0–6.5) –
EDSS: expanded disability status scale; RRMS: relapsing–remitting multiple sclerosis; SPMS: secondary progressive multiple sclerosis; PPMS: primary progressive multiple sclerosis; HC: healthy controls. a Data are expressed as mean (SD). b Data are expressed as median (interquartile range).
(SPMS, n = 39), or primary progressive (PPMS, n = 73) according to the Lublin and Reingold classification (Lublin and Reingold, 1996).
3. Results 3.1. Serum sTNF-RII levels are elevated in patients with PPMS
To investigate a potential relationship between serum sTNF-RII levels and disability progression, a subgroup of PPMS patients (n = 24) was classified according to worsening of disability over a two-year period. Disability progression was defined as a confirmed increase in the EDSS score of at least 1 point if initial EDSS score was lower than 5.5, and of at least 0.5 points if initial EDSS score was equal or higher than 5.5 during the follow-up period. Serum levels of sTNF-RII were measured at baseline and after two years of follow-up in PPMS patients with disability progression (n = 11) and without disability progression (n = 13). 2.4. Determination of serum levels of sTNF-RII by enzyme-linked immunosorbent assay Peripheral blood was collected by standard venipuncture and serum was prepared after centrifugation of the clotted blood and stored frozen at − 80 °C until used. Serum levels of sTNF-RII were determined by a sandwich enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Human sTNF RII/TNFRSF1B Immunoassay, R&D), according the manufacturer's protocol. Samples were diluted at a 1:50 ratio by dilution buffer and added to ELISA plates to measure the sTNF-RII content in the serum. Plates were analyzed at OD 450 nm using an ELx800 Absorbance Microplate Reader (BioTek, VT, USA) and calculated using Gen5 Data Analysis Software (BioTek, VT, USA). All assays were performed in duplicate and samples with variation coefficients higher than 10% were repeated. The intra-assay and inter-assay coefficients of variation were 2.5% and 10% respectively. 2.5. Statistical analysis Statistical analysis was performed by using the SPSS 17.0 package (SPSS Inc, Chicago, IL) for MS-Windows. The ANOVA test was first used to analyze differences in serum sTNF-RII levels between MS patients with different clinical forms and healthy controls. An unpaired Student's t test was then used to test for significant differences in serum sTNF-RII levels between PPMS patients, MS patients with other clinical forms of the disease (RRMS and SPMS), and healthy controls. A paired Student's t-test was used to test for significant differences in serum sTNF-RII levels between PPMS patients at baseline and after two years of follow-up. Linear associations between serum sTNF-RII levels and clinical and radiological variables were assessed by the Pearson or Spearman rank correlation coefficients depending on the applicability conditions. Descriptive data are presented as mean values [standard deviation (SD)] unless otherwise stated.
Serum sTNF-RII levels were analyzed in serum samples from healthy controls and MS patients with different clinical forms of MS. As shown in Fig. 1, serum sTNF-RII levels were significantly increased in PPMS patients compared with RRMS patients (p = 0.005), SPMS patients (p = 0.047) and healthy controls (p = 0.018). 3.2. Levels of serum sTNF-RII are not associated with disability progression in PPMS patients We next investigated whether serum sTNF-RII levels changed over time in a subgroup of 24 PPMS patients. As shown in Fig. 2A, comparisons of sTNF-RII levels over the 24-month follow-up period revealed a statistically significant increase in protein levels at 24 months versus the baseline (p = 3.8 × 10−7). We further analyzed whether changes in serum sTNF-RII during the follow-up period could discriminate between patients with and without disability progression. Demographic
p=0.018 p=0.005 5000
p=0.047
4000
sTNF-RII (pg/ml)
2.3. Serum sTNF-RII levels and disability progression
3000
2000
1000
0 HC
RR
(N=46)
(N=49)
SP
(N=39)
PP
(N=73)
Fig. 1. Comparison of serum sTNF-RII levels between MS patients with different clinical forms of the disease and healthy controls. Box plots showing serum sTNF-RII levels in MS and healthy controls. sTNF-RII levels were determined by ELISA as described in Materials and methods. The levels of the soluble receptor are increased in PPMS patients compared with RRMS patients (median values: 2928 pg/ml in PPMS vs. 2594 pg/ml in RRMS; p = 0.005), SPMS patients (median: 2731 pg/ml; p = 0.047) and healthy controls (median: 2661 pg/ml; p = 0.018). Boxes represent the 90th and 10th percentiles divided horizontally by the median. Whiskers are drawn to the nearest value not beyond a standard span from the 90th and 10th percentiles. RR: patients with relapsing–remitting MS. PP: patients with primary progressive MS. SP: patients with secondary progressive MS. HC: healthy controls.
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N. Fissolo et al. / Journal of Neuroimmunology 271 (2014) 56–59
A
4. Discussion
7000
p=3.8x10-7
sTNF-RII (pg/ml)
6000 5000 4000 3000 2000 1000 0
B
7000
0
24
(N=24)
(N=24)
p=4.2x10-4
p=4.9x10-4
sTNF-RII (pg/ml)
6000 5000 4000 3000 2000 1000 0 24
0
no progression (N=13)
0
24
progression (N=11)
Fig. 2. Changes in serum sTNF-RII levels over a 24-month follow-up in PPMS patients. Box plots showing the serum sTNF-RII levels in PPMS patients (n = 24) at baseline and after 24 months of follow-up. sTNF-RII levels were determined by ELISA. (A) Levels of sTNF-RII are increased over time in the whole group of PPMS patients (p = 3.8 × 10−7). (B) In PPMS patients classified by disability progression, sTNF-RII levels were significantly elevated in both groups of patients (p = 4.9 × 10−4 in patients with progression and p = 4.2 × 10−4 in patients without progression). Boxes represent the 90th and 10th percentiles divided horizontally by the median. Whiskers are drawn to the nearest value not beyond a standard span from the 90th and 10th percentiles.
The present study was designed to investigate the serum levels of one of the soluble receptors of TNF-α, sTNF-RII, in different clinical forms of MS. Our results show a selective increase of sTNF-RII levels in PPMS patients compared with patients with other clinical forms of the disease and healthy controls. In addition, we observed that in this group of patients sTNF-RII levels tended to increase over time, although this finding was irrespective of disability progression. Finally, in PPMS patients we observed a lack of association between radiological variables, measuring inflammation and atrophy, and serum levels of sTNF-RII probably indicating that the levels of this soluble receptor may not reflect the whole extent of CNS damage. Studies over the past years suggest that soluble TNF receptors may enhance or inhibit TNF activity depending on their concentration (Aderka et al., 1992; Mohler et al., 1993). The pro-inflammatory cytokine TNF-α has been associated with MS disease activity and implicated in axon degeneration (Rieckmann et al., 1995; Siebert and Bruck, 2003). On the other hand, TNF-α has also potent anti-inflammatory properties (Liu et al., 1998), and several studies using anti-TNF therapy in MS patients have shown to increase disease activity (The Lenercept Multiple Sclerosis Study Group, 1999). These pro-inflammatory and antiinflammatory properties of TNF-α make it difficult to hypothesize whether changes in serum levels of sTNF-RII will be beneficial or detrimental in MS. In our study, the selective increase in sTNF-RII serum levels in PPMS patients suggests a role of the receptor in this particular progressive form of MS, since sTNF-RII levels were not elevated in the more inflammatory but also progressive form of SPMS. However, as outlined above, sTNF-RII did not correlate with disability progression or radiological measures of atrophy. Based on these findings, further studies will be required to elucidate the precise role of sTNF-RII in patients with PPMS. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.jneuroim.2014.04.001. Acknowledgments The authors thank the “Red Española de Esclerosis Múltiple (REEM)” sponsored by the FEDER-FIS and the “Ajuts per donar Suport als Grups de Recerca de Catalunya”, sponsored by the “Agència de Gestió d'Ajuts Universitaris i de Recerca” (AGAUR), Generalitat de Catalunya, Spain. References
and baseline clinical characteristics were comparable between both groups of patients (Supplementary Table 1). As illustrated in Fig. 2B, serum sTNF-RII levels were similarly and significantly increased in PPMS patients with disability progression (p = 4.9 × 10− 4) and patients without disability progression (p = 4.2 × 10−4).
3.3. Correlations between serum sTNF-RII levels and clinical and radiological variables No significant correlations were found between serum TNF-RII levels in patients with different clinical forms of MS and clinical variables such as disease duration, number of relapses in the previous 2 years, and EDSS score at the time of blood drawing (correlation coefficient range: from −0.144 to 0.251). Similarly, no statistically significant correlations were observed between sTNF-RII levels in PPMS patients and baseline radiological variables such as T2-weighted lesion load, T1-weighted lesion load, brain parenchymal fraction, and spinal cord area (correlation coefficient range: 0.018–0.090).
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