Elevated Osteopontin Levels in Active Relapsing-Remitting Multiple Sclerosis Mario H. J. Vogt, PhD,1 Luba Lopatinskaya, MD,1,2 Monique Smits, BSc,1 Chris H. Polman, MD, PhD,2 and Lex Nagelkerken, PhD1 In the search for proteins that might play a role in the pathogenesis of multiple sclerosis (MS), osteopontin (OPN) has been identified as the most prominent cytokine-encoding gene expressed within MS lesions. Here, we report significantly increased OPN protein levels in plasma of relapsing-remitting MS patients. In contrast, OPN protein levels in primary progressive and secondary progressive MS patients were similar to healthy control levels. Interestingly, active relapsing-remitting patients had higher OPN protein levels than patients without relapses. Ann Neurol 2003;53:819 – 822 Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system.1,2 High-throughput sequencing of cDNA from MS brain lesions and control brains has showed that osteopontin (OPN) is the most abundant cytokine-encoding transcript unique to MS plaques.3 MS tissues have been probed immunohistochemically for the expression of OPN, and its level was particularly elevated in reactive astrocytes and microglial cells.3 OPN is a negatively charged acidic hydrophilic protein of 314 amino acids and has pleiotropic functions, including a role in both acute and chronic inflammation.4 OPN is involved in the recruitment of macrophages and T cells to inflamed sites.5,6 An interaction between the C-terminal domain of OPN and the receptor CD44 expressed by macrophages induces chemotaxis, and engagement of ␤3-integrin receptors by the N-terminal OPN domain induces activation.7 Interestingly, mice deficient in OPN gene expression (OPN⫺/⫺) have severely impaired type 1 immunity to viral and bacterial infections.6 OPN⫺/⫺ mice were also resistant to progressive EAE and had frequent remis- From the 1Division of Immunological and Infectious Diseases, TNO Prevention and Health, Leiden; and 2Department of Neurology, VU Medical Center, Amsterdam, The Netherlands. Received Feb 7, 2003, and in revised form Mar 7. Accepted for publication Mar 18, 2003. Address correspondence to Dr Vogt, Division of Immunological and Infectious Diseases, TNO Prevention and Health, P.O. Box 2215, 2301 CE, Leiden, The Netherlands. E-mail: firstname.lastname@example.org sions, indicating that OPN plays an important role in the demyelination process.3,8 Cells from draining lymph nodes from OPN⫺/⫺ mice demonstrated impaired interleukin (IL)–12 and IFN␥ production and increased IL-10 production compared with OPN⫹/⫹ mice, suggesting that OPN is a T helper cell 1 (Th1) cytokine. In view of the functional role of OPN and its ubiquitous tissue expression, we determined whether OPN protein levels in serum of MS patients were associated with disease activity. Subjects and Methods Patients and Controls Thirty relapsing-remitting (RR), 10 secondary progressive (SP), and 10 primary progressive (PP) MS patients at the Department of Neurology, VU Medical Center, Amsterdam were included in this study for determination of OPN protein baseline levels. Ten healthy volunteers were included as a control group. The demographic characteristics of the MS patients are given in the Table. Relapse was defined as the appearance or reappearance of one or more neurological abnormalities that persisted for at least 24 hours, and which had been preceded by at least 30 days of stable or improved neurological state. Twenty of these RR MS patients were selected for a prospective immunological and clinical follow-up over the course of 6 months immediately after initiation of interferon (IFN)–␤1b treatment. Treatment consisted of subcutaneous injections of 8 million international units of IFN-␤1b on alternate days. EDTA blood was collected at 0 (baseline) and 1, 3, and 6 months after initiation of the therapy. Enzyme-linked Immunosorbent Assay The OPN content in EDTA plasma was measured using a standard ELISA according to the manufacturer’s procedure (Assay Designs, Ann Arbor, MI). Data Analysis All statistical tests were performed using the MS Windows SPSS 11.0 package. One-way analysis of variance followed by a post hoc analysis (Bonferroni) was used for group comparisons in the cross-sectional approach. For all serial samples, the analysis of variance module (univariate general linear model) was used. In the model, the subjects were taken as a random factor, and different time points were included as fixed factors. Once a significant difference among the time groups was found, the Bonferroni post hoc test was used to determine which pairs of groups differed. Reported p values were two tailed and p value less than 0.05 was considered significant in this exploratory study. Results Baseline Osteopontin Protein Levels in Multiple Sclerosis Patients and Controls OPN protein levels in serum of MS patients with different clinical subtypes were evaluated and compared © 2003 Wiley-Liss, Inc. 819 Table. Demographic and Clinical Characteristics of Multiple Sclerosis Patients Clinical Subtypes No. of patients Gender (female to male ratio) Mean age (yr ⫾ SD) Disease duration (yr ⫾ SD) EDSS at intake (mean ⫾ SD) PP RR SP 10 6:4 51.2 ⫾ 12.6 14.0 ⫾ 9.8 6.1 ⫾ 1.6 30 25:5 39 ⫾ 6.2 7.8 ⫾ 5.3 3.6 ⫾ 1.5 10 5:5 47.4 ⫾ 6.9 12.2 ⫾ 7.7 4.9 ⫾ 1.1 PP ⫽ primary progressive; RR ⫽ relapsing remitting; SP ⫽ secondary progressive; SD ⫽ standard deviation. EDSS ⫽ expanded disability status scale. with levels present in healthy controls. As depicted in Figure 1, the median OPN concentration in 10 healthy controls was 175ng/ml, and the levels showed only little variation between subjects. PP MS patients had a slightly increased median value of 224ng/ml, demonstrating high variation of OPN levels between subjects. However, the OPN levels in the PP MS group did not significantly differ from levels in healthy controls. In contrast with the PP MS group, OPN levels in the RR MS group were significantly ( p ⬍ 0.001) enhanced by 60% compared with the levels in healthy controls. OPN levels in SP patients were comparable to levels in healthy controls. Clinical Exacerbations and Osteopontin Protein Levels in Plasma of Relapsing-remitting Multiple Sclerosis Patients The OPN protein levels in the RR MS patient group displayed a rather large variation between subjects. Because these patients develop repeated clinical exacerbations followed by remissions, the variance of Fig 1. Osteopontin (OPN) protein levels in plasma of healthy controls (HCs), primary progressive (PP), relapsing-remitting (RR), and secondary progressive (SP) multiple sclerosis (MS) patients. OPN protein concentration in EDTA plasma of each subject was determined using a standard enzyme-linked immunosorbent assay. The line represents the median value of each patient group. RR MS patients had significantly elevated OPN protein levels compared with the healthy controls (Bonferroni’s post hoc after one-way analysis of variance, p ⬍ 0.001). 820 Annals of Neurology Vol 53 No 6 June 2003 OPN levels may be the result of differences in disease activity at the time of sampling. Out of the 30 abovementioned RR patients, 20 received treatment with IFN-␤1b, and their disease activity was very well documented both before and after start of treatment. A total of 13 relapses were noticed in 11 of these patients during the time period from 3 months before until 3 months after sampling of plasma for OPN protein baseline evaluation. OPN protein baseline levels were significantly ( p ⫽ 0.019) enhanced in these 11 patients who developed relapses as compared with the nine patients in which no relapse was noticed during this interval (Fig 2). In fact, OPN levels in the latter group were not significantly ( p ⫽ 0.218) different from the levels observed in the healthy control group. Fig 2. Elevated osteopontin (OPN) protein levels in plasma of relapsing-remitting multiple sclerosis patients who developed relapses. OPN protein concentrations in EDTA plasma of each subject was determined using a standard enzyme-linked immunosorbent assay. During the time period of 3 months before until 3 months after sampling, 13 relapses were noticed in 11 of 20 patients. Those patients who developed relapses had significantly (p ⫽ 0.019) higher OPN protein baseline levels compared with the nine patients for whom no relapse was noticed during this observation period. The line represents the median value of each patient subgroup. Effect of Interferon-␤1b Treatment on Osteopontin Protein Levels in Relapsing-remitting Multiple Sclerosis Patients To determine whether IFN-␤1b treatment had an effect on the OPN protein levels in the RR MS patients, the OPN plasma concentration in each patient was measured before initiation of IFN-␤1b treatment and compared with the levels after 1, 3, and 6 months of treatment. The effect of IFN-␤1b on OPN protein levels is depicted as percentage difference from OPN baseline levels (Fig 3). Surprisingly, OPN protein levels were slightly but significantly ( p ⬍ 0.001) enhanced after 1 month of IFN-␤1b treatment, with a 15% increase in median value compared with baseline samples (T0). However, after 3 and 6 months of IFN-␤1b therapy OPN levels declined to baseline levels again. Discussion In the search for immunomodulatory proteins that are important in MS, microarray analysis and highthroughput sequencing of cDNAs from MS brain lesions showed that the level of transcripts for OPN was elevated.3 Because OPN is a ubiquitously expressed protein, OPN upregulation in various cell types may result in elevated plasma levels of this protein in MS patients. In this study, we demonstrated that RR MS patients had significantly increased OPN levels, whereas PP and SP MS patients had similar OPN levels as healthy controls. The OPN protein levels in the RR MS patient group displayed a rather large variation between subjects. We showed that the Fig 3. Effect of recombinant interferon (IFN)–␤1b treatment on osteopontin (OPN) protein levels in plasma of relapsingremitting MS patients. OPN protein concentrations in EDTA plasma of each subject was determined using a standard ELISA. The OPN protein level in each subject was measured at initiation of IFN-␤1b treatment (OPN T0) and after 1 (OPN T1), 3 (OPN T3) and 6 months (OPN T6) of treatment. The percentage differences were calculated as (OPN Tx ⫺ OPN T0)/OPN T0*100. OPN protein levels were slightly but significantly upregulated during the first month of IFN-␤1b treatment (p ⬍ 0.001), followed by a decline to baseline from 3 months onward. variance of OPN levels was the result of differences in disease activity in each subject at the time of sampling. Patients with high disease activity had higher OPN protein levels than patients without clinical exacerbations. OPN has pleiotropic functions, including a role as Th1 cytokine with the ability to increase IFN-␥ and IL-12 production and to decrease IL-10 release by immune cells.6 Previously, we demonstrated that peripheral blood mononuclear cells in MS patients have increased levels of IL-12p40 and decreased levels of IL-10 mRNA as compared with controls.9 Elevated expression of the IL-12p40 subunit has been found in the cerebrospinal fluid of RR patients with active plaques, and MS patients had increased numbers of IFN-␥–secreting cells in peripheral blood and cerebrospinal fluid compared with controls.10 These observations indicate that the Th1/Th2 balance is dysregulated. Increased levels of OPN in serum of RR MS patients may contribute to this phenomenon. IFN-␤ treatment of RR patients reduces relapse rate by approximately 30%.11 The mechanism by which IFN-␤ works remains unclear, although several possible mechanisms have been suggested. IFN-␤ may inhibit the entry of peripheral blood mononuclear cells into the central nervous system by downregulation of adhesion molecules on brain endothelial cells and by antagonizing increased migration of peripheral blood mononuclear cells in response to chemokines.12,13 IFN-␤ may also have immunomodulatory effects on different cell subsets and their cytokine production.14 For example, IFN-␤ treatment enhances IL-10 mRNA and protein levels and increases the percentage of IL10 –secreting peripheral blood T lymphocytes.15,16 Surprisingly, we found slightly but significantly enhanced OPN protein levels in plasma of RR patients during the first month of IFN-␤1b treatment. Studies indicate that the OPN promoter contains various motifs including IFN-inducible elements.17,18 These elements may be responsible for the initial OPN upregulation within the first month of IFN-␤1b therapy. This finding is in line with other studies, which showed that IFN-␤ is able to induce Th1 markers.19 Moreover, it has been demonstrated that IFN-␤1b treatment induces elevation of IFN-␥–producing cell numbers in MS patients during the first 2 months of treatment, followed by a normalization after 3 months.20 Similar effects of IFN␤1b on OPN protein levels have been observed in this study. In conclusion, increased OPN protein levels in plasma of RR MS patients are associated with disease activity. These increased levels may contribute to a dysregulation of the Th1/Th2 balance as has been suggested by several studies. Vogt et al: Osteopontin Levels in MS 821 This work was supported by the Dutch Foundation for the Support of MS Research (Stichting Vrienden MS Research). We are grateful for Drs S. Dijkstra and H. van Noort for critically reviewing the manuscript. References 1. Steinman L, Martin R, Bernard C, et al. 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