Journal of the Neurological Sciences 295 (2010) 31–37

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Journal of the Neurological Sciences

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Dysregulated neurotrophin mRNA production by immune cells of patients withrelapsing remitting multiple sclerosis

Nataly Urshansky a, Karin Mausner-Fainberg b, Eitan Auriel a,b, Keren Regev a,Firas Farhum a, Arnon Karni a,b,⁎a Neuroimmunology Laboratory, Department of Neurology, Tel Aviv Sourasky Medical Center, Israelb Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel

⁎ Corresponding author. Department of Neurology, Te6Weizmann Street, Tel Aviv 64239, Israel. Tel.: +972 3 6

E-mail address: arnonk@tasmc.health.gov.il (A. Karn

0022-510X/$ – see front matter © 2010 Elsevier B.V. Adoi:10.1016/j.jns.2010.05.019

a b s t r a c t

a r t i c l e i n f o

Article history:Received 19 March 2010Received in revised form 26 April 2010Accepted 18 May 2010Available online 11 June 2010

Keywords:Multiple sclerosisNeurotrophinsmRNACytokines

Recent studies have suggested a neuroprotective activity of the lesion-infiltrating immune cells in multiplesclerosis (MS) by secretion of neurotrophins. We had earlier reported that immune cells from relapsingremitting MS (RR-MS) patients secrete low levels of brain-derived neurotrophic factor (BDNF), and that itssecretion is dysregulated after CD40 stimulation. Here, we measured mRNA levels for BDNF, NT3 and NGF-βmRNA in unstimulated PBMCs and found levels lower in untreated RR-MS patients than in healthy controls(HC). T-cell stimulation with anti-CD3/CD28 mAb up-regulated neurotrophin mRNA expression in untreatedRR-MS patients and not in HC, whereas stimulation of PBMCs with anti-CD40 mAb up-regulatedneurotrophin mRNA expression in HC and not in RR-MS patients. Further cellular analyses of the productionof the neurotrophin mRNA in individual cells revealed that T cells were the main producers of theneurotrophin mRNA in RR-MS patients, and that monocytes were the main producers of NT3 and NGF-βmRNA in HC. BDNF mRNA was similarly produced in monocytes and T cells in the HC group. The cytokinesTNF-α and IL-17 up-regulated the expression of neurotrophin mRNA in HC but not in RR-MS patients. Theneuroprotective activity of PBMCs appeared to be dysregulated in untreated patients with RR-MS, while thedifferences between the IFN-β-treated RR-MS patients and the HC were smaller.

l Aviv SouraskyMedical Center,973424; fax:+972 3 6974872.i).

ll rights reserved.

© 2010 Elsevier B.V. All rights reserved.

1. Introduction

Multiple sclerosis (MS) is themost common disabling neurologicaldisease of young adults [1]. The majority (85%) of patients with MSinitially have a relapsing remitting disease (RR-MS) course charac-terized by clearly defined alternating episodes of neurologic deteri-oration, variable extent of recovery and considerable length ofremission between the attacks [2].

This disease is characterized by inflammatory cell infiltration ofthe CNS white matter, demyelination and axonal damage [3–6]. Theextent of the patient's disability correlates with the neuronal loss[7,8]. The inflammatory mechanisms contribute to axonal pathologyand demyelination, but probably also have some neuroprotectiveproperties mediated by the release of neurotrophic factors [9–14].Neurotrophins are a group of structurally related proteins that includethe following growth factors: brain-derived neurotrophic factor(BDNF), neurotrophin-3 (NT3) and nerve growth factor (NGF).These proteins are able to prevent neural death, favor the recoveryprocess, and contribute to neural regeneration and remyelination

[15]. BDNF was found to promote the survival and differentiation ofneurons, which naturally express it in the brain [10]. Several studieshave shown that the therapeutic application of BDNF preventsneuronal degeneration after experimental axotomy and other formsof neuronal injury [16]. BDNF secretion by genetically engineeredbone marrow stem cells reduced the clinical severity, the inflamma-tion and the apoptosis in experimentally induced autoimmuneencephalomyelitis (EAE) in a murine model of MS [17]. NGF-βpromoted themyelin synthesis by oligodendrocytes in the CNS and bySchwann cells in the peripheral nervous system [18,19] to induce theexpression of early neuronal markers and the differentiation ofoligodendrocytes by cells of the subventricular zone in the EAE[20,21]. NGF-β inhibited monocyte migration through the activatedblood-brain barrier (BBB) in a culture model of the endothelial BBB[22]. NT3 promoted proliferation and survival of oligodendrocytes[23]. BDNF, NGF-β and NT3 can be expressed and secreted within theCNS by immune system cells, such as activated T lymphocytes andmonocytes [10,11,24]. Activated Th1 cells accumulating in the CNShave the potential to disrupt axons and kill neurons [25]. Theinflammatory process may, however, also have beneficial effects: itenhanced axonal survival and could even improve functionality inexperimental models of optic nerve crush and spinal trauma [26].Myelin-reactive T cells demonstrated neuroprotective effects in EAE[11,27], and activated immune cells produced neurotrophic factors

32 N. Urshansky et al. / Journal of the Neurological Sciences 295 (2010) 31–37

[28]. Finally, the perivascular infiltrate in humanMS brain lesions wasshown to contain BDNF, NT-3, and NGF [29].

A significant number of BDNF-containing immune cells weredetected in actively demyelinating areas of human MS lesions [10].Thus, BDNF and other neurotrophic factorsmay be capable ofmediatinga neuroprotective effect of the immune system in MS [13,24]. We hadpreviously reported that immune cells from patients with relapsingremittingMS (RR-MS) secrete lower levels of BDNF thanHC and that itssecretion is dysregulated after CD40 stimulation [30,31]. In the currentstudy on a completely different set of patients and HC, we investigatedthe expression and regulation of mRNA of the neurotrophins, BDNF,NGF and NT3 in the immune cells of RR-MS patients, in order explorethe cause of the lower secretion of BDNF in RR-MS compared to HC, andto study the other neurotrophins in RR-MS compared to HC.

2. Methods

2.1. Patients and controls

Patients with definite MS who were attending the Neuroimmu-nology Clinic at the Tel Aviv SouraskyMedical Center were included inthe study. A definitive diagnosis of MS was made according to thecriteria of McDonald et al. [32]. Blood samples were drawn from 50patients with RR-MS in clinical remission for at least 3 months (21untreated and 29 treated with interferon-β) and from 38 age-matched HC. No patient had any other chronic inflammatorycondition, and none of the participants were being treated with otherimmune/inflammatory-related medication (e.g., NSAIDS, steroids orother immunosuppressive agents). Table 1 describes the characteristicsof the study participants. Due to the limited amounts of RNA that wereproduced, the exact number of participants for each experiment ismentioned with the results.

All experiments were approved by the institutional ethicscommittee and informed consent was obtained from all participants.

2.2. Separation of peripheral blood mononuclear cells

Peripheral blood mononuclear cells (PBMCs) were isolated fromheparinized venous blood by centrifugation over Ficoll-Paque(Amersham Biosciences, Uppsala, Sweden). The cells were resus-pended (2×106 cells/ml) in complete culture medium comprised ofRPMI 1640 medium supplemented with 10% fetal bovine serum,4mM L-glutamine, 25 mM Hepes buffer, penicillin 50 units/ml, andstreptomycin 50 μg/ml (Biological Industries, Kibbutz Bet Haemek,Israel).

Table 1Clinical characteristics of the participants.

Untreatedpatients

IFN-β-treatedpatientsa

Healthycontrols

Number 21 29 38Males 8 10 14Females 13 19 24Age (mean±SD), years 33.7±6.8 36.1±7.1 34.6±6.4Range, years (19–48) (20–53) (22–45)EDSS score (mean±SD) 1.9±1.5 2.3±1.7Range (0–4.0) (0–4.5)Disease duration, years 5.1±2.1 5.7±3.2Range, years (1–9) (1–13)

EDSS, Expanded Disability Standard Scale.a 8 were treated once a week with intramuscular IFN-β1a, 13 were treated 3 times a

week with subcutaneous IFN-β1a and 8 were treated with subcutaneous IFN-β1b everyother day.

2.3. T cells and monocytes separation

Purification of monocytes (CD14+) and T cells (CD3+ cells) fromPBMCswas performed by a positive selection of CD14+ cells followingan additional positive selection of CD3+ cells using a magnet-activated cell separation extraction kit (Miltenyi Biotec Inc., BergischGladbach, Germany). Cell purity was determined by flow cytometryusing PE-conjugated mouse monoclonal antibody (mAb) anti-humanCD3, FITC-conjugated mouse mAb anti-human CD14 and theircorresponding isotype controls. The purity was N95% for the CD3+

and CD14+ populations (all from BD Biosciences Pharmingen, SanDiego, CA, USA).

2.4. PBMC culture conditions

PBMCs at a concentration of 2.5×106 cells/ml were either notstimulated or they were stimulated with soluble 1 μg/ml mouse anti-human CD3 monoclonal Ab (mAb) and soluble 5 μg/ml mouse anti-human CD28 mAb (BD Biosciences Pharmingen) or soluble10 μg/mlmouse anti-human CD40 mAb and their isotype controls (IC) (R&DSystem, Minneapolis, MN, USA) for 1 h and then collected for totalRNA isolation. The timing of RNA collection was determined after atime-course experiment we did in which neurotrophin mRNA wasdetected after 30 min, 1 h, 2 h, 4 h, 8 h and 24 h. Themaximal levels ofneurotrophin mRNA for both stimulation with CD3/CD28 and CD40were detected after 1 h. PBMCs (2.5×106 cells/ml) were alsoincubated with either 100 ng/ml of either human IL-10 or IL-17 orINF-γ or TNF-α or without exogenous cytokine for 1 h and thenproceeded to total RNA isolation (all cytokines from R&D System). Theconcentrations of cytokines were defined according to our previousexperiments [31].

2.5. Real-time quantitative RT-PCR of BDNF mRNA

Total RNA was prepared from PBMCs using an Easy RNApurification kit (Biological Industries) according to themanufacturer'sinstructions. The total RNA samples were routinely treated withTurbo-DNase (Ambion) to prevent possible genomic DNA contami-nation. Before studying the real-time qRT-PCR reactions, the absenceof any residual genomic DNA contamination was confirmed by usingPCR reaction with GAPDH primers of samples that did not undergo invitro transcription reaction. The total RNA of 1 μg was transcribedwith random hexamers using Reverse iT transcriptase (Verso cDNAkit) following themanufacturer's instructions. Real-time qRT-PCRwasperformed on an ABI Prism 7900HT Instrument (Applied Bioscience).BDNF expression was tested by real-time qRT-PCR at 94 °C for 10 minand then by 45 cycles as follows: 94 °C for 15 s, 60 °C for 15 s, and72 °C for 15 s. The melting curve analysis was routinely used for eachreaction. The GAPDH gene was run in parallel for internal control foreach reaction set. GAPDH expression was independent of any PBMCtreatment (data not shown). The oligonucleotide primer sequencesused were:

BDNF forward primer 5′-CCAGGGACCTCTCTCTAATCAG-3′ andBDNF reverse primer 5′-CCTGGGAGTAGATGAGGTACAG-3′,GAPDH forward primer 5′-ACCACAGTCCATGCCATCAC-3′ andGAPDH reverse primer 5′-TCCACCACCTGTTGCTGTA-3′.NT3 forward primer 5′-AAAAAGGTTGCAGGGGTAT-3′NT3 reverse primer 5′-TTGGGATGTTTTGCACTGAG-3′NGF forward primer 5′-GAA CCA CAC TCA GAG AGC AAT GTC-3′NGF reverse primer 5′-AGT GTC AAG GGA ATG CTG AAG TTT-3′.

2.6. Statistical analysis

The data were calculated as relative quantification values accord-ing to the ABI PRISM 7900HT software and normalized against GAPDH±SEM. p values were calculated by Student's t test and corrected for

Fig. 1. The basal level of neurotrophin expression in untreated RR-MS patients was lowerthan in healthy controls (HC). Total RNA from PBMCs was isolated from the studyparticipants and the level of neurotrophin mRNA expression was studied by real-timePCR. (A) BDNF mRNA was lower in untreated RR-MS patients than in HC. The levels inIFN-β-treated patients tended to be higher than in the untreated patients but withoutstatistical significance. (B) NT3mRNA levelswere lower in both untreated RR-MS patientsand IFN-β-treated patients compared to HC, and no statistical differences were foundbetween the patients groups. (C) NGF-β mRNA was lower in the untreated RR-MSpatients compared to HC. No statistical differences were found between the patientgroups. Data are presented as relative quantification values normalized against GAPDH±SEM.

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multiple comparisons where needed by multiplying them by thenumber of comparisons.

3. Results

3.1. PBMCs of RR-MS patients expressed lower levels of neurotrophinsmRNA compared to HC

We first measured the ex-vivo expression of neurotrophins inPBMCs with no stimulation. Data are presented as relative quantifi-cation values normalized against GAPDH±S.E.M. In the untreatedgroup of patients the levels of BDNF mRNA (1.57±0.54, n=19), NT3mRNA (1.74±0.45, n=21) and NGF-β mRNA (1.51±0.44, n=18)were lower than in the HC group (BDNF mRNA=2.86±0.39, n=36,p=0.045; NT3 mRNA=3.46±0.58, n=38, p=0.023; NGF-βmRNA=3.22±0.49, n=35, p=0.013). The expression levels ofneurotrophins mRNA in the IFN-β treated patients were: BDNFmRNA=1.86±0.53, n=26; NT3 mRNA=1.71±0.42, n=29; NGF-βmRNA=2.23±0.55, n=27. There were no statistical differencesbetween these levels and those of the untreated patient group (Fig. 1).

3.2. Stimulation of PBMCs via CD3/CD28 increased the expression ofneurotrophins mRNA in untreated RR-MS patients

We then tested the effect of stimulation via CD3/CD28 on theneurotrophin expression levels in RR-MS patients compared to HC. TheBDNF mRNA levels in untreated RR-MS patients (n=17) after 24 h ofincubation with soluble anti CD3/CD28 mAb were 6.40±3.08 com-pared to incubation with isotype controls (IC: 1.58±0.54, p=0.036),while there were no statistical differences between the incubation withanti-CD3/CD28 mAb (2.63±0.74) and with IC (2.93±0.40) in the HCgroup (n=26). The NT3 mRNA levels in untreated RR-MS patients(n=19) after incubation with anti-CD3/CD28 mAb were 5.94±2.15compared to incubation with IC (1.26±0.31, p=0.039), while therewere no statistical differences between the incubation with anti-CD3/CD28 mAb (2.11±0.56) and with IC (2.08±0.44) in the HC group(n=25). The NGF-βmRNA levels in untreated RR-MS patients (n=16)after incubation with anti-CD3/CD28 mAb were 6.02±2.07 comparedto incubation with IC (1.23±0.41, p=0.028), while there were nostatistical differences between incubation with anti-CD3/CD28 mAb(2.22±0.62) and with IC (2.74±0.54) in the HC group (n=24). Thiseffect of stimulation with CD3/CD28 was smaller in the IFN-β-treatedgroup: BDNF mRNA after incubation with anti-CD3/CD28 mAb (2.21±0.16) compared to incubation with IC (1.86±0.53, p=N.S), n=24,NT3 mRNA after incubation with anti-CD3/CD28 mAb (2.93±1.41)compared to incubation with IC (0.92±0.30, p=0.034), n=16, andNGF-βmRNAmRNA after incubation with anti-CD3/CD28mAb (6.03±1.73) compared to incubation with IC (3.36±1.16, p=0.046), n=19(Fig. 2).

3.3. Stimulation of PBMCS via CD40 failed to up-regulate neurotrophinexpression in RR-MS patients

We had previously shown that stimulation with anti-CD40antibody failed to up-regulate the secreted levels of BDNF proteinfrom RR-MS patients compared to HC [30,31]. Here, we studied theeffect of stimulation via CD40 on the mRNA expression of theneurotrophins, BDNF, NGF and NT3.

The BDNF mRNA levels in untreated RR-MS patients (n=19) after24 h of incubation with soluble anti-CD40 mAb were (1.59±0.54)compared to the incubation with IC (1.84±0.81, p=N.S), whileincubationwith anti-CD40mAb up-regulated BDNFmRNA expression(6.83±1.87) compared to IC (2.45±0.49, p=0.028) in the HC group(n=24). The NT3 mRNA levels in untreated RR-MS patients (n=19)after incubation with anti-CD40 mAb were 2.45±1.32 compared to1.61±0.74 for incubation with IC (p=NS), while incubation with

Fig. 2. Neurotrophin expression was increased in CD3/CD28-stimulated PBMCs of RR-MS patients. We analyzed the expression levels of BDNF, NT3 and NGF-β mRNA after 1 h ofstimulation with soluble anti-human CD3/CD28 antibody or with their isotype control. The levels of neurotrophins mRNA were detected by real-time PCR with the appropriateprimers. (A) BDNF mRNA levels in untreated RR-MS patients incubation with soluble anti-CD3/CD28 mAb were significantly higher than the incubation with isotype controls (IC),while there was no differences between the two incubation conditions. (B) The NT3 mRNA levels in untreated RR-MS patients after incubation with anti-CD3/CD28 mAb weresignificantly higher than with IC, while there was no statistical difference between the two incubation conditions in the HC group. (C) The NGF-β mRNA levels in untreated RR-MSpatients after incubation with anti-CD3/CD28mAbwere significantly higher thanwith IC, while there were no statistical differences between the two incubation conditions in the HCgroup. In the IFN-β-treated group: BDNF mRNA after incubation with anti-CD3/CD28 mAb was similar to incubation with IC. NT3 mRNA levels after stimulation with anti-CD3/CD28mAb were significantly higher than with IC and NGF-β mRNA after incubation with anti CD3/CD28 mAb was significantly higher than with IC. The figure presents the changebetween the two conditions (stimulation via CD3/CD28 and IC) in each participant and it is expressed as a relative quantification value normalized against GAPDH±SEM.

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anti-CD40 mAb up-regulated its expression (12.52±3.12) comparedto IC (3.19±1.15, p=0.033) in the HC group (n=24). The NGF-βmRNA levels in untreated RR-MS patients (n=18) after incubationwith anti-CD40 mAb were 0.95±0.26 compared to 1.46±0.44 forincubation with IC (p=NS), while incubation with anti-CD40 mAbup-regulated its expression (10.53±2.87) compared to incubationwith IC (3.89±2.13, p=0.049) in the HC group (n=24).

The effect of stimulation via CD40 in the IFN-β-treated group wasas follows: BDNF mRNA after incubation with anti-CD40 mAb was2.84±0.43 compared to 2.08±0.63 for IC (p=N.S) n=24; NT3mRNA after incubation with anti-CD40 mAb was 1.66±0.48 com-pared to 1.43±0.38 for incubation with IC (p=N.S), n=24; NGF-βmRNA mRNA after incubation with anti-CD40 mAb was 3.35±0.67compared to 2.16±0.58 for incubation with IC (p=0.049), n=25(Fig. 3).

3.4. Dissimilarity between untreated RR-MS patients and HC inneurotrophin mRNA expression in T cells and monocytes

We reasoned that the differences in the responses to stimulations byCD3/CD28 and CD40 between untreated RR-MS patients and HCmay beattributed to dissimilar expressions of neurotrophinmRNAs in differentcell populations, since CD3/CD28 is most frequently expressed on Tcells and CD40 is most frequently expressed on monocytes among thePBMCs. To test this possibility, we separated T lymphocytes (CD3+),monocytes (CD14+) and other cells (“rest”) from the PBMCs of HC(n=7) and of RR-MS patients (n=7) and studied the expression ofneurotrophin mRNA in these populations. The HC group showed asimilar expression level of BDNF mRNA in CD3+ cells (10.18±1.36)compared to CD14+ cells (11.41±2.48, p=N.S). However, the NT3

mRNA expression was lower in CD3+ cells (7.49±2.46) compared toCD14+ cells (14.42±2.84, p=0.044), and NGF-β mRNA expressionwas lower in CD3+ cells (3.42±1.06) compared to CD14+ cells (6.49±1.43, p=0.048), while the expression level of BDNF mRNA was higherin CD3+ cells (2.48±1.00) compared to CD14+ cells (0.23±0.05,p=0.044) in RR-MS patients. Similarly, NT3 mRNA expression washigher in CD3+ cells (10.94±2.76) compared to CD14+ cells (2.77±0.89, p=0.039), and NGF-βmRNA expressionwas higher in CD3+ cells(4.90±1.88) compared to CD14+ cells (1.10±0.48, p=0.046) in RR-MS patients. Comparing the patient groups to the HC group, BDNFmRNA expression in the CD3+ cells was significantly higher in HCcompared to the levels of expression in the RR-MS patients (p=0.002).No significant differences were found for NT3 and NGF-βmRNAs. In theCD14+ cells, BDNF mRNA expression was higher in HC compared tothe RR-MS patients (p=0.011), NT3 mRNA expression was higher inHC compared to the RR-MS patients (p=0.044), and NGF-β mRNAexpression was higher in HC compared to the RR-MS patients(p=0.038).

The “rest” cells were never dominant producers of any of theneurotrophin mRNA. In HC, BDNF mRNA was 5.88±1.71, NT3 mRNAwas 4.09±1.60 and NGF-βmRNAwas 0.69±0.20, while these valueswere 1.99±0.58, 6.17±2.65 and 1.61±0.81, respectively, in un-treated RR-MS patients (Fig. 4).

3.5. TNF-a and IL-17 failed to up-regulate neurotrophins mRNAexpression in RR-MS

Cytokines, the chemical messengers between immune cells, playcrucial roles in mediating inflammatory and immune responses. Wetested the influence of cytokines that are associated with the function

Fig. 3. Total RNA was isolated from PBMCs that were stimulated for 24 h with either soluble mouse anti-human CD40 mAb (CD40) or its isotype control (IC). The levels ofneurotrophins mRNA were detected by real-time PCR with the appropriate primers. (A) The BDNF mRNA levels in untreated RR-MS patient incubation with soluble anti-CD40 mAbwere similar to the levels with ICs. In the HC group, however, incubation with anti-CD40 mAb significantly up-regulated the BDNF mRNA expression compared to IC. The NT3 mRNAlevels in untreated RR-MS patients after incubation with anti-CD40 mAb were similar to the levels with IC, while incubation with anti-CD40 mAb significantly up-regulated its levelscompared to IC in the HC group. The NGF-β mRNA levels in untreated RR-MS patients after incubation with anti-CD40 mAb were similar to the levels with IC, while the incubationwith anti-CD40 mAb significantly up-regulated it compared to IC in the HC group. The effect of stimulation via CD40 in the IFN-β-treated group was as follows: BDNF mRNA afterincubation with anti-CD40 mAb tended to be higher after stimulation via CD40 compared to IC but without statistical differences. NT3 mRNA after incubation with anti-CD40 mAbwas similar to incubation with IC. NGF-β mRNA after incubation with anti-CD40 mAb was significantly higher than with IC. The figure presents the change between the twoconditions (stimulation via CD40 and IC) in each participant and it is expressed as a relative quantification value normalized against GAPDH±SEM.

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of Th1, Th17 and regulatory cells on the expression of neurotrophicfactors in untreated patients with RR-MS compared to HC (Fig. 5). Ourdata demonstrated significant increases in all three neurotrophicfactor expressions by TNF-α only in HC group. The BDNF mRNA ratio(TNF-α/no cytokine) was 0.67±0.23 in RR-MS patients (n=12)compared to 5.61±2.16 in HC (n=11, p=0.049). The NT3 mRNAratio (TNF-α/no cytokine) ratio was 1.09±0.03 in RR-MS patients(n=11) compared to 12.41±4.29 in HC (n=10, p=0.029). TheNGF-β mRNA ratio (TNF-α/no cytokine) was 1.45±0.41 in RR-MSpatients (n=11) compared to 14.38±5.34 in HC (n=11, p=0.038).Interleukin 17, the product of Th17 cells, increased the expression ofneurotrophic factors mRNA in HC. The BDNF mRNA ratio (IL17/no

Fig. 4. Neurotrophins are expressed differently in each cell population among healthy conisolated from T lymphocytes (CD3+), monocytes (CD14+) and other cells (“rest”) populatiexpression of GAPDH mRNA. Monocytes produced higher levels of neurotrophin mRNA inneurotrophin mRNA in untreated RR-MS patients. The levels of BDNFmRNA from T lymphocyRR-MS.

cytokine) was 1.27±0.26 in RR-MS patients (n=12) compared to2.49±0.49 in HC (n=11, p=0.027). The NT3 mRNA ratio (IL17/nocytokine) was 1.56±0.42, in RR-MS patients (n=11) compared to3.43±0.72 in HC (n=11, p=0.042). The NGF-β mRNA ratio (IL17/nocytokine) was 1.22±0.23 in RR-MS patients (n=11) compared to3.84±1.10 in HC (n=11, p=0.049).

INF-γ, a Th1 cytokine, tended to increase the expression of the threeneurotrophin mRNAs in both RR-MS patients and HC except for BDNFmRNA. The BDNF mRNA ratio (INF-γ/no cytokine) was 0.65±0.22 inRR-MS patients (n=11) compared to 3.78±1.42 in HC (n=11,p=0.048). The NT3 mRNA ratio (INF-γ/no cytokine) was 4.03±1.27inRR-MS patients (n=11) compared to 7.16±4.22, n=11 in HC

trols (HC) and untreated RR-MS patients. Real-time PCR was performed on total RNAons. The values of expression of each neurotrophin mRNA were normalized against theHC compared T lymphocytes and the “rest”, while T cells were the main producers oftes of HC, however, were higher than those of T lymphocytes of untreated patients with

Fig. 5. The effect of TNF-α, IL-17, IFN-γ and IL-10 on the expression of neurotrophins mRNA BDNF, NT3 and NGF-β mRNA expression. Real-time PCR was performed on total RNAisolated from PBMCs stimulated by one of these cytokines. The analysis of mRNA expression levels of BDNF, NT3 and NGF-β showed significant differences in neurotrophin mRNAexpression between healthy individuals (HC) and patients with RR-MS, especially after exposure to TNF-α and IL-17. Data are presented as average of the ratios between the levels ofexpression of mRNA in the presence of exogenous cytokine compared to the levels without exogenous cytokine. ⁎p≤0.05

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(p=NS). The NGF-β mRNA ratio (INF-γ/no cytokine) was 5.46±1.96in RR-MS patients (n=11) compared to 5.60±1.87 in HC (n=11,p=NS). IL-10, an immunoregulatory cytokine, showed an effect onlyon the HC group. The BDNF mRNA ratio (IL10/no cytokine) was 1.32±0.73 in RR-MS (n=11) compared to 2.96±1.35 in HC (n=11, p=NS).The NT3 mRNA ratio (IL10/no cytokine) was 0.47±0.09 in RR-MS(n=10) compared to 3.71±1.08 in HC (n=11, p=0.031). The NGF-βmRNA ratio (IL10/no cytokine) was 1.43±0.13in RR-MS (n=11)compared to 5.08±1.40 in HC (n=11, p=0.048).

4. Discussion

Inflammatory responses may have neuroprotective capacity viathe production of neurotrophins. We had earlier reported that theability of immune cells to produce BDNF is reduced and dysregulatedin patients with RR-MS [31]. We now report that basal expressionlevels of neurotrophin mRNA in patients with RR-MS were lowercompared to those of healthy individuals. BDNF, NT3 and NGF-βmRNA levels in HC were expressed mostly in monocytes, whereastheir expression was found preferentially in T lymphocytes in RR-MSpatients. However, with regard to BDNF mRNA production in CD3+

cells, HC produced higher levels than the patients with RR-MS. Wealso found that stimulation via CD3/CD28 up-regulated neurotrophinmRNA expression in RR-MS patients but not in HC. This finding couldbe attributed to a shift in the expression of these factors towards Tcells in RR-MS patients. Our previous data showed an increase ofBDNF protein secretion after CD40 pathway activation in healthyindividuals but not in RR-MS patients [30,31]. Here again, thestimulation of PBMSc via CD40 demonstrated an increase in theexpression of all three tested neurotrophin mRNAs in HC but not inuntreated RR-MS patients.

We had previously shown that the secreted levels of BDNF fromPBMCs of RR-MS patients treated with IFN-β1a were up-regulated viathe stimulatory CD40 pathway like they had been in the HC group [31].In the current study on a different group of IFN-β1a-treated RR-MS

patients, we found intermediate features of neurotrophins mRNAexpression between those of untreated RR-MS patients and those ofHC. Therewas a trend for elevation of BDNFmRNAbasal levels and afterstimulation via CD40, and a significant up-regulation of NGF-β mRNAafter stimulation via CD40 (like that seen in HC), and an up-regulatoryeffect of NT3 and NGF-βmRNAs following stimulation with CD3/CD28.

Several studies have shown that pro-inflammatory cytokines, suchas TNF-α, are elevated in serum and/or cerebrospinal fluids of MSpatients, and that these levels are often correlated with diseaseprogression [33,34]. Production of the regulatory cytokine, IL-10, wasassociated with less disability [35], and its up-regulation wasobserved in patients that were treated with β-interferon [36,37].Interleukin-17 has been reported to be over-produced in EAE, as wellas in patientswithMS [38,39].We demonstrated that TNF-α and IL-17increase the expression of neurotrophinmRNA in HC but not in RR-MSpatients. IFN-γ increased the expression of all three neurotrophinmRNAs, of NT3 mRNA and of NGF-β mRNA, but not of BDNF mRNA inboth HC and RR-MS patients. IL-10 increased the expression of NT3mRNA and NGF-β mRNA in HC but not in RR-MS patients.

Our results show that the production of neurotrophin mRNA bythe immune cells of patients with RR-MS is dysregulated. The overallproduction in PBMCs is low in untreated RR-MS patients, and there isa cellular shift from monocytes to T cells in the dominant producer.The monocytes of RR-MS patients lose their capacity to produceneurotrophin mRNA, while, on the other hand, the patient's T cells aremore sensitive to CD3/CD28 stimulation than the T cells of HC.However, the stimulation of PBMCs of HC via CD40 was stronger thanthe stimulation of PBMCs of untreated RR-MS patients via CD3/CD28,especially for NT3 and NGF-β mRNAs.

In summary, PBMCs of patients with RR-MS produce fewerneurotrophin mRNAs than HC, a feature which is probably relatedto a defective response of monocytes to stimuli of CD40 and ofimmune cells to TNF-α IL-17 and IL-10 that play a role in the immuneresponse of MS. This reduced and altered neurotrophin mRNAproduction in the PBMCs of RR-MS patients may be related to a

37N. Urshansky et al. / Journal of the Neurological Sciences 295 (2010) 31–37

failure in neuroprotection activity by the immune cells in the disease.It would be interesting to study neurotrophin expression in otherchronic inflammatory conditions as well, in order to verify if these lowand dysregulated expressions of neurotrophins are specific forpatients with RR-MS or if they also occur in patients with otherinflammatory diseases. The altered nonspecific immunity of cells inpatients with MS, such as monocytes, dendritic cells and otherantigen-presenting cells, has been reported previously [39–42], andour current work adds new data on this abnormal immunologicalphenomenon in patients with MS.

Acknowledgment

Esther Eshkol, the institutional medical copy editor of Tel AvivSourasky Medical Center, is thanked for editorial assistance.

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